CRYSTALLOGRAPHIC STUDIES INARI ON THE STRUCTURE-FUNCTION KURSULA RELATIONSHIPS IN Biocenter Oulu and Department of Chemistry, TRIOSEPHOSPHATE ISOMERASE University of Oulu

OULU 2003

INARI KURSULA

CRYSTALLOGRAPHIC STUDIES ON THE STRUCTURE-FUNCTION RELATIONSHIPS IN TRIOSEPHOSPHATE ISOMERASE

Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on May 16th, 2003, at 12 noon.

OULUN YLIOPISTO, OULU 2003 Copyright © 2003 University of Oulu, 2003

Supervised by Professor Rik Wierenga

Reviewed by Doctor Kristina Djinovic Carugo PhD Mikael Peräkylä

ISBN 951-42-7009-6 (URL: http://herkules.oulu.fi/isbn9514270096/)

ALSO AVAILABLE IN PRINTED FORMAT Acta Univ. Oul. A 400, 2003 ISBN 951-42-7008-8 ISSN 0355-3191 (URL: http://herkules.oulu.fi/issn03553191/)

OULU UNIVERSITY PRESS OULU 2003 Kursula, Inari, Crystallographic studies on the structure-function relationships in triosephosphate isomerase Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu; Department of Chemistry, University of Oulu, P.O.Box 3000, FIN-90014 University of Oulu Oulu, Finland 2003

Abstract The triosephosphate isomerase (TIM) barrel superfamily is a broad family of proteins, most of which are enzymes. At the amino-acid-sequence level, many of the members of this family share little, if βα any, homology. Yet, they adopt the same three-dimensional ( )8 fold. The TIM barrel fold seems to be a good framework for many different kinds of enzymes, providing unique possibilities for both natural and human-designed evolution, as the catalytic center and the stabilizing features are separated to different ends of the barrel. Indeed, in the light of most recent studies, it seems likely that at least most of the different TIM barrel enzymes, catalyzing a huge variety of reactions, have evolved from a common ancestor. TIM can be considered a real text-book enzyme — its catalytic properties and stucture-function relationships have been studied for decades. Still, at present, we are quite far from understanding the structural features that make TIM and other enzymes such superior catalysts in both efficiency and precision. TIM is a dimeric enzyme that consists of two identical subunits of 250 residues. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in glycolysis. The basics of this reaction are well known, but there is ongoing discussion about the details of the proton transfer steps, and three alternative pathways have been suggested. In addition, it is a fascinating question how the enzyme succeeds in abstracting a highly stable proton from a carbon atom of the substrate. This study was undertaken to shed light on some of the questions concerning the structure- function relationships in TIM. The most important findings are the elucidation of the role of Asn11 as a catalytic residue and the meaning of the flexibility of both the catalytic Glu167 side chain as well as the substrate during catalysis, and the presence of a low-barrier hydrogen bond between Glu167 and a transition-state analogue, 2-phosphoglycolate. Furthermore, significant results were obtained on the importance of a conserved salt bridge, 20 Å away from the active site and the dimer interface, for the stability and folding of TIM as well as on the factors influencing the opening of the flexible loop 6 upon product release.

Keywords: catalysis, enzyme, low-barrier hydrogen bond, protein folding, proton abstraction

For Petri, Ville, Lotta, and Jussi

Acknowledgements

This work was carried out at the Department of Biochemistry and Biocenter Oulu, University of Oulu, during the years 1998-2003. I owe deep gratitude to professor Rik Wierenga for his expertise and most kind supervision. I am also grateful to professor Kalervo Hiltunen and all the other professors, group leaders, and staff at the department for creating excellent working conditions. I would like to thank Dr. Kristina Djinovic and Dr. Mikael Peräkylä for their critical review of this manuscript and helpful suggestions, and Eliisa Kursula for careful revision of the language. I thank all my co-authors for their contributions, especially Dr. Annemie Lambeir for instructive discussions on enzyme kinetics. I wish to thank all the nice and helpful people at the synchrotrons I have visited - EMBL/DESY, ESRF, and MAX-Lab. I am grateful to all former and present members of the RW group for helping in many practical and theoretical matters, and for being such good company. Especially Anu, Gitte, Mira, Päivi, and Sanna have ensured that the days at work have not been all too serios, but also lots of fun. During these years, I have not had enough time to spend with friends. Still, I could always count on them being there. Besides being great company, Kati, Kaisa, Mika, Taru, and Katri have often been available for baby sitting. Jarmo and Sanna, despite living far from us, have meant a lot for our family. I am most grateful for the friendship of Kati and Petri, and their children Jaakko and Anni. I owe my warmest thanks to my parents, who always encouraged me to ask questions and raised the curiosity in me. I am also grateful to my little sister Taru for numerous moments of joy. Yet more, I want to thank my parents-in-law for helping whenever needed. Most of all people, I would like to thank my dear Petri for his love and support. Besides my husband and best friend, he has been my best teacher and collaborator. Our three wonderful children, Ville, Lotta, and Jussi, are the joy of my life and have always helped me remember what life really is about. This work was financially supported by the Finnish Academy and Suomalainen Konkordia-liitto.

Oulu, May 2003 Inari Kursula

Abbreviations

ADP adenosine 5’-monophosphate ATP adenosine 5’-triphosphate C carboxyl CD circular dichroism DGAP D-glyceraldehyde-3-phosphate DHAP dihydroxyacetone phosphate E65Q the mutant Leishmania mexicana mexicana TIM, where Glu65 is replaced by Gln E. coli Escherichia coli FMNH reduced form of flavin adenine mononucleotide G3P glycerol-3-phosphate HisA N-[(5’-phosphoribosyl)formimino]-5-aminoimidazole-4- carboxamide ribonucleotide isomerase HisF imidazole glycerol phosphate synthase IPP 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate kDa kilodalton LBHB low-barrier hydrogen bond L. mexicana Leishmania mexicana mexicana N amino NAD oxidized form of nicotinamide adenine dinucleotide NADH reduced form of nicotinamide adenine dinucleotide NADP nicotinamide adenine dinucleotide phosphate NMR nuclear magnetic resonance PDB Protein Data Bank 3PG 3-phosphoglycolate PGA 2-phosphoglycolate PGH phosphoglycolohydroxamate Pi inorganic phosphate P. woesei Pyrococcus woesei Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis T. brucei Trypanosoma brucei brucei TIM triosephosphate isomerase TLS translation, libration, screw rotation T. maritima Thermatoga maritima Tris tris(hydroxymethyl)aminomethane TrpA the a -subunit of tryptophan synthase TrpC indole-3-glycerol phosphate synthase TrpF phosphoribosylanthranilate isomerase V. marinus Vibrio marinus YGGS the Tyr210-Gly211-Gly212-Ser213 region in loop 7 of TIM List of original articles

This thesis is based on the following articles, referred to in the text by their Roman numerals.

I Lambeir AM, Backmann J, Ruiz-Sanz J, Filimonov V, Nielsen JE, Kursula I, Norledge BV & Wierenga RK (2000) The ionisation of Glu65 buried at the monomer-monomer interface in Leishmana mexicana triosephosphate isomerase is thermodynamically linked to the stability of the dimer. Eur J Biochem 267: 2516-24.

II Kursula I, Partanen S, Lambeir AM, Antonov DM, Augustyns K & Wierenga RK (2001) Structural determinants for ligand binding and catalysis of triosephosphate isomerase. Eur J Biochem 268: 5189-5196.

III Kursula I, Partanen S, Lambeir AM & Wierenga RK (2002) The importance of the conserved Arg191-Asp227 salt bridge of triosephosphate isomerase for folding, stability, and catalysis. FEBS Lett 518: 39-42.

IV Kursula I & Wierenga RK (2003) Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-Å resolution. J Biol Chem 278: 9544-9551.

Contents

Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction...... 15 2 Review of the literature...... 16 2.1 The TIM barrel superfamily of proteins...... 16 2.1.1 The TIM barrel structure...... 16 2.1.2 Classification of TIM barrel proteins...... 20 2.1.3 Evolution of TIM barrel proteins...... 22 2.1.3.1 Convergent evolution towards a stable fold?...... 23 2.1.3.2 Evidence for divergent evolution from a common ancestor.... 24 2.1.3.3 TIM barrels in different genomes...... 26 2.1.4 Folding and stability of TIM barrel proteins...... 26 2.1.5 Engineering the TIM barrel fold...... 28 2.2 Triosephosphate isomerase...... 28 2.2.1 Structural features...... 29 2.2.1.1 The overall structure...... 29 2.2.1.2 Folding and stability...... 29 2.2.1.3 The dimer interface...... 30 2.2.1.4 Active-site construction...... 30 2.2.2 The gene, expression, and cellular localization of triosephosphate isomerase...... 34 2.2.3 The function of triosephosphate isomerase...... 34 2.2.3.1 Glycolysis...... 34 2.2.3.2 The catalytic mechanism...... 36 2.2.3.3 Substrate and transition-state analogues...... 40 2.2.3.4 Kinetic properties...... 42 2.2.4 Triosephosphate isomerase deficiency...... 43 2.2.5 Protein engineering studies on triosephosophate isomerase...... 45 3 Aims of the present study...... 46 4 Materials and methods...... 47 4.1 Recombinant protein expression and purification...... 47 4.2 Site-directed mutagenesis and characterization of the salt bridge mutant proteins...... 47 4.3 Crystallization...... 48 4.4 X-ray diffraction data collection and processing...... 48 4.5 Structure determination, refinement, and validation...... 49 4.5.1 Molecular replacement...... 49 4.5.2 Refinement of the medium-resolution structures...... 49 4.5.3 Refinement of the atomic-resolution structure...... 49 4.6 Structural comparisons...... 51 5 Results...... 52 5.1 Structure of the Leishmania mexicana triosephosphate isomerase superstable E65Q mutant...... 52 5.2 Comparison of the binding mode of 2-(N-formyl-N-hydroxy)- aminoethyl phosphonate and phosphoglycolohydroxamate...... 52 5.3 Mutagenesis and structural studies on the role of the conserved salt bridge between Arg191 and Asp227...... 54 5.4 The atomic-resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate...... 55 5.4.1 Quality of the model...... 55 5.4.2 Structural heterogeneity...... 56 5.4.3 Anisotropy...... 57 5.4.4 Active-site geometry...... 58 6 Discussion...... 60 6.1 The role of the conserved salt bridge Arg191-Asp227 in folding and stability...... 60 6.2 Superstability of the E65Q mutant...... 61 6.3 The active site...... 63 6.3.1 The catalytic role of Asn11...... 63 6.3.2 Adaptability of the active site and the possible role of low-barrier hydrogen bonds in the catalytic mechanism...... 63 6.3.3 Loop 6 and loop 7...... 67 7 Concluding remarks...... 70 References 1 Introduction

Enzymes are vital for virtually all processes in living cells. They can speed up reactions that otherwise would be far too slow to have any significance for life. The rate enhancement by enzymes can be up to 1017-fold (Radzicka & Wolfenden 1995). This enormous facilitation of reactions is largely due to the ability of enzymes to stabilize the transition state better than the substrate and product. Many of the most efficient enzymes belong to the superfamily of triosephosphate isomerase (TIM) barrel proteins. This is the most common fold among proteins, and most of the family members are enzymes. It is estimated that even ten percent of all proteins share this fold. TIM barrel enzymes catalyze a variety of reactions, but almost all of them are related to key metabolic pathways. Because so abundant, the TIM barrel fold is of great interest for evolutionary research, as well as for protein design studies. A wealth of information on the structure and evolutionary relationships of this class of proteins is now available. In this literature review, the current knowledge on TIM barrel structure and evolution is summarized. In addition to an extensive amount of research articles, several excellent review articles have recently been published on these topics (Pujadas & Palau 1999, Wierenga 2001, Pujadas 2002). TIM is an enzyme that catalyzes the interconversion between dihydroxy- acetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) in glycolysis (Knowles & Albery 1977, Knowles 1991). The structure and function of TIM have been studied extensively for decades, especially since the determination of its three-dimensional structure (Banner et al. 1975). Still, the detailed mechanism of catalysis is unclear, and many questions concerning the structure-function relationships remain unanswered. In the literature part of this thesis, various aspects of TIM are addressed based on some of the extensive literature available. This study was initiated to find answers to some of the questions concerning conserved structural features in TIM and their effects on the function of this enzyme. As the work was carried out, more emphasis was put on understanding the exact steps of the catalytic mechanism and the structural details of the active- site that make TIM such an efficient catalyst. 2 Review of the literature

2.1 The TIM barrel superfamily of proteins

2.1.1 The TIM barrel structure

The TIM barrel fold, first discovered in chicken (Gallus gallus) TIM (Banner et al. 1975) (Fig. 1), is one of the most common folds among proteins - possibly even ten percent of all proteins belong to this class, most of them being enzymes. Sequence identity within the members of this family is rather low, but the structural similarity is high enough to define the (ba )8 fold as the canonical TIM barrel. One TIM barrel domain has typically approximately 250 residues. TIM barrel proteins can be monomers or even tetramers of these, or they can be multi-domain proteins, in which the TIM barrel domain is just one part of a bigger complex. The TIM barrel fold is characterized by eight parallel b strands that are hydrogen bonded to each other forming a barrel-like structure in the core of the protein, surrounded by eight a helices connected to the preceeding strands by loop regions of varying length. The a helices cover the b strands such that the hydrophobic face is towards the b sheet and the hydrophilic face interacts with solvent. The ba loops (the loops connecting a b strand to the following a helix) tend to be longer than the ab loops. Sometimes these loops are large enough to form separate domains, as, for example, in pyruvate kinase (Larsen et al. 1994) and a -amylase (Song et al. 1996). In both of these, loop3 has been replaced by a separate domain. The b strands are numbered as strand 1-8 from the amino terminus to the carboxy terminus, and the following a helices similarily as helix 1-8 (Fig. 2). Some of the proteins regarded as TIM barrels deviate significantly from the canonical barrel structure (Pujadas & Palau 1999) (Fig. 3). To give a few examples, in enolase, one of the b strands is antiparallel to the rest of the b barrel (Lebioda et al. 1989, Wedekind et al. 1994), whereas in nicotinate-nucleotide pyrophosphorylase, one of the strands is missing (Eads et al. 1997). Also some of the a helices can be missing, as in endo-b-N-acetylglucosaminidase (Rao et al. 17

1995). Cellulases, flavoprotein 390, and quinolinic acid phosphoribosyltransferase only have seven b strands, there is also one nine- stranded member in the phosphatidylinositole phospholipase family. The TIM barrel domain of methylmalonyl-CoA mutase closes up to form the barrel structure only when the substrate is bound (Mancia & Evans 1998). One half is formed from ba units 6, 7, 8, and 1, and the other from ba units 2-5.

A B

Fig. 1. Three-dimensional structure of chicken TIM (PDB entry 1TPH; Zhang et al. 1994a). A) Viewing along the barrel axis. The active site at the C-terminal end of the b strands is facing to the front. B) Rotated 90° about the horizontal axis from the previous view, showing the side of the barrel. The active site is at the top. The figure was generated using Molscript (Kraulis 1991).

N C 1 2 3 4 5 6 7 8

Fig. 2. Topology diagram of the TIM barrel fold. The b strands are depicted as arrows and the a helices as cylinders. The loops at the bottom of the figure are the N-terminal loops and the loops at the top are the C-terminal loops. 18

A B

C D

Fig. 3. Some TIM barrel proteins that significantly deviate from the canonical fold. A) One of the central b strands is missing in nicotinate-nucleotide pyrophosphorylase (1QAP; Eads et al. 1997). B) Methylmalonyl-CoA mutase (1REQ; Mancia et al. 1996) closes up to form a TIM barrel structure upon substrate binding. C) In enolase (1EBG; Wedekind et al. 1994), one of the central b strands is antiparallel compared to the rest of them. D) a -amylase (1VJS; Song et al. 1996.) has one intercalated domain. The figure was generated using Molscript (Kraulis 1991).

The b strands are connected to each other via main chain hydrogen bonds between neighboring strands. Despite being called a barrel, the core structure most often is not circular but rather elliptical. The length of the b strands varies from short, as in TIM (Banner et al. 1975), to long, as in xylose isomerase (Farber et al. 1987). The barrel structure is stabilized by layers of mostly hydrophobic side chains pointing both towards the center of the barrel as well as the space between the b strands and the surrounding a helices (Fig. 4). There are normally three of these layers, each of them consisting of four residues from alternating strands (reviewed in Wierenga 2001, Pujadas & Palau 1999). The pattern of residues within the same layer, pointing either towards or away from the interior of the barrel, causes the TIM barrel to have fourfold symmetry, instead of eightfold (Nagano et al. 1999, Pujadas & Palau 1999). 19

Fig. 4. Schematic representation of the b strands forming the core of the TIM barrel of Trypanosoma brucei brucei TIM. The circles refer to residues pointing inside the barrel (closed circles) and the space between the b strands and a helices (open circles). The shear number (S) is 8, meaning that the b strands are tilted such that moving around the barrel along corresponding residues, the end point is displaced by 8 residues with respect to the starting point. The tilt of the b strands with respect to the barrel axis is approximately 35°. Layers 1, 2, and 3 refer to the contact layers inside the barrel. The dotted lines indicate hydrogen-bonding interactions. The figure was reproduced, with permission, from Wierenga (2001).

Most of the a helices in TIM barrel proteins are amphipathic and packed over the corresponding b strands leaving a very hydrophobic interface between the b barrel and the surrounding a helices. Indeed, the most hydrophobic region in most TIM barrel proteins is this interface instead of the inside of the barrel (Nagano et al. 1999), which, in some cases, like the enolase barrel, can be even hydrophilic (Babbitt et al. 1996). The N-terminal loops in TIM barrel proteins show, in general, the least sequence similarity compared to the rest of the protein. As the active site is situated at the carboxyl (C) terminal end of the b barrel, the amino (N) terminal loops are far from the active site. It has been postulated that these loops have an important role in stabilizing the overall structure, as large insertions in the case of phosphoribosylanthranilate isomerase (TrpF) are only possible within the C- terminal loops, at the expense of catalytic activity, rather than the N-terminal loops (Urfer & Kirschner 1992). C-terminal loops form the active site and are, thus, important for the catalytic properties of TIM barrel enzymes. In addition, these loops stabilize the 20 quarternary structures that most TIM barrel proteins form (Borchert et al. 1994). For example, in TIM, the dimer is formed as residues from the tip of loop 3 are buried between loop 1 and loop 4 in the dimer interface (Borchert et al. 1994). In general, the C-terminal loops tend to be longer than the N-terminal ones. In some cases, these loops are long enough to form separate domains, as for example in pyruvate kinase (Larsen et al. 1994). The active sites of all TIM barrel enzymes are located at the C-terminal ends of the b strands and are shaped by the ba loops (Farber & Petsko 1990, Pujadas & Palau 1999). Yet, the catalytic residues come from different locations along the sequence (Nagano et al. 1999). The substrates of TIM barrel enzymes are mostly negatively charged, allowing for a favourable interaction with the electrostatic field generated by the main chain and side chains of the TIM barrel. This electrostatic field is positive near the active site (Raychaudhuri et al. 1997). In fact, many of the substrates have a phosphate moiety. The actual phosphate- binding sites occur most often in the C-terminal part of the sequence (loops 7 and 8), and in many of the TIM barrel proteins, there exists a short phosphate- binding helix inserted in loop 8 (Wilmanns et al. 1991). A number of TIM barrel enzymes bind cofactors, such as flavin mononucleotide, nicotinamide adenine dinucleotide phosphate (NADP), pyridoxal-5’-phosphate, or divalent metals (Nagano et al. 2002). In the enzymes using cofactors, they are always bound in the C-terminal part of the b barrel, like the substrates. Metal ions are often bound in equivalent sites in different TIM barrel enzymes (Nagano et al. 2002).

2.1.2 Classification of TIM barrel proteins

As the TIM barrel undoubtedly is the most common fold adopted by enzymes, it is clear that very many different types of enzymes bear this fold. In the 1999 release of CATH (Class, Architecture, Topology, and Homologous superfamily) (Orengo et al. 1997), TIM barrel proteins were divided into 18 superfamilies of homologous structures including 889 TIM barrel folds in 503 Protein Data Bank (PDB) entries (Nagano et al. 1999). In the current Structural Classification of Proteins (SCOP) database, 25 TIM barrel superfamilies based on structural similarity can be found. On the other hand, according to their enzyme activities, TIM barrel proteins can be divided into six classes, five of which are actually enzymes (Pujadas & Palau 1999) (Table 1). It can be noted that of the six enzyme classes according to the Enzyme Commission (E.C.), ligases are the only group in which no TIM barrel enzymes have been found. Most of the known TIM barrel enzymes are hydrolases. Others include oxidoreductases, lyases, isomerases, and transferases. Only a handful of TIM barrel proteins have no or an unknown enzyme activity (Table 1). By far most of the TIM barrel proteins of known structure are involved in energy, macromolecule or small molecule metabolism (Rison et al. 2000). 21

Table 1. Classification of TIM barrel proteins based on enzyme activities according to the TIM barrel database (Pujadas & Palau 1999). ______Class E.C. No. of Name of the protein code PDB entries ______Oxidoreductases 1.1.1.- 1 2,5-diketo-D-gluconic acid reductase A 1.1.1.2 3 alcohol dehydrogenase (NADP+(1)) 1.1.1.21 14 aldehyde reductase 1.1.1.50 2 3-a -hydroxysteroid dehydrogenase (B-specific) 1.1.1.205 3 inosine monophosphate dehydrogenase 1.1.1.213 1 3-a -hydroxysteroid dehydrogenase (A-specific) 1.1.2.3 5 L-lactate dehydrogenase (cytochrome) 1.1.3.15 4 (S)-2-hydroxy-acid oxidase 1.3.3.1 2 dihydroorotate oxidase 1.5.99.7 1 trimethylamine dehydrogenase 1.6.99.1 5 NADPH(2) dehydrogenase 1.7.99.5 1 5,10-methylenetetrahydrofolate reductase (FADH(3)) 1.14.14.3 4 alkanal monooxygenase (flavin mononucleotide-linked) Transferases 2.2.1.2 2 transaldolase 2.4.1.19 29 cyclomaltodextrin glucanotransferase 2.4.2.19 1 nicotinate-nucleotide pyrophosphorylase (carboxylating) 2.4.2.29 4 queuine transfer ribonucleic acid ribosyltransferase 2.5.1.3 1 thiamin-phosphate pyrophosphorylase 2.5.1.15 5 dihydropteroate synthase 2.7.1.40 9 pyruvate kinase 2.7.9.1 3 pyruvate,phosphate dikinase Hydrolases 3.1.4.10 11 1-phosphatidylinositol phosphodiesterase 3.1.4.11 11 1-phosphatidylinositol-4,5-bisphosphate phospho- diesterase 3.1.8.1 3 aryldialkylphosphatase 3.1.21.2 2 deoxyribonuclease IV (phage T4-induced) 3.2.1.1 28 a -amylase 3.2.1.2 9 b-amylase 3.2.1.4 15 cellulase 3.2.1.8 15 endo-1,4-b-xylanase 3.2.1.10 1 oligo-1,6-glucosidase 3.2.1.14 1 chitinase 3.2.1.21 4 b-glucosidase 3.2.1.23 4 b-galactosidase 3.2.1.31 1 b-glucuronidase 3.2.1.39 1 glucan endo-1,3-b-D-glucosidase 3.2.1.52 4 b-N-acetylhexosamidinase 3.2.1.60 5 glucan 1,4-a -maltotetrahydrolase 3.2.1.68 1 isoamylase 3.2.1.73 2 licheninase 3.2.1.78 3 mannan endo-1,4-b-mannosidase 3.2.1.85 4 6-phospho-b-galactosidase 3.2.1.91 9 cellulose 1,4-b-cellobiosidase ______22

Table 1. Continued ______Class E.C. No. of Name of the protein code PDB entries ______3.2.1.96 2 mannosyl-glycoprotein endo-b-N-acetylglucosamidase 3.2.1.135 1 neopullulanase 3.2.3.1 2 thioglucosidase 3.5.1.5 21 urease 3.5.4.4 8 adenosine deaminase Lyases 4.1.1.31 1 phosphoenolpyruvate carboxylase 4.1.1.39 20 ribulose-bisphosphate carboxylase 4.1.1.48 5 indole-3-glycerol-phosphate synthase 4.1.2.13 8 fructose-bisphosphate aldolase 4.1.2.14 1 2-dehydro-3-deoxyphosphogluconate aldolase 4.1.2.15 1 2-dehydro-3-deoxyphosphoheptonate aldolase 4.1.3.3 3 N-acetylneuraminate lyase 4.2.1.11 13 phosphopyruvate hydratase 4.2.1.20 13 tryptophan synthase 4.2.1.24 2 porphobilinogen synthase 4.2.1.40 1 glucarate dehydratase 4.2.1.52 1 dihydrodipicolinate synthase Isomerases 5.1.1.1 3 alanine racemase 5.1.2.2 6 mandelate racemase 5.1.3.1 1 ribulose-phosphate 3-epimerase 5.3.1.1 39 triosephosphate isomerase 5.3.1.5 63 xylose isomerase 5.3.1.24 2 phosphoribosylanthranilate isomerase 5.4.2.9 1 phosphoenolpyruvate mutase 5.4.99.2 7 methylmalonyl-coenzyme A mutase 5.5.1.1 4 muconate cycloisomerase 5.5.1.7 2 chloromuconate cycloisomerase Unknown enzyme activity 1 concanavalin B 1 narbonin 2 nonfluorescent flavoprotein 1 phosphotriesterase homology protein 2 yeast hypothetical protein ______(1)oxidized nicotinamide adenine dinucleotide phosphate (2)reduced nicotinamide adenine dinucleotide phosphate (3)reduced flavin adenine dinucleotide ______

2.1.3 Evolution of TIM barrel proteins

The evolution of TIM barrel proteins has been subject to intense studies since late 1970s (Levine et al. 1978). It has been stated that such a diverse class of enzymes, in the sense of both sequence and function, possessing the same three- dimensional fold could have evolved either by convergent evolution from 23 several unrelated ancestors or by divergent evolution from one or very few ancestors. During the last few years, the interest towards the evolutionary relationships of TIM barrel proteins has evermore increased. The development of new methods, and, especially, the availability of structural information on many different TIM barrel enzymes, has given valuable new insights into the question of TIM barrel evolution (reviewed by Wierenga 2001, Pujadas & Palau 1999, Pujadas 2002).

2.1.3.1 Convergent evolution towards a stable fold?

The model of convergent evolution in the case of TIM barrel proteins was first proposed by Hilary Muirhead in 1983. This theory was based on the low sequence homology between the known members of this family and the apparent eightfold symmetry of the superimposed structures that were used (Muirhead 1983). The reason for several unrelated proteins ending up with the same three-dimensional structure would be the very high stability of such a fold. When the active sites of TIM and muconate lactonizing enzyme are superimposed, it is noted that b strands 1 and 3, respectively, are homologous. This has been considered as evidence supporting the theory of convergent evolution (Goldman et al. 1987). Later, another example was considered to be enolase (Lebioda et al. 1989) which looks like a TIM barrel protein, but the second b strand and the first a helix are antiparallel compared to the rest of the strands and helices. The side chain interactions inside the b barrels of TIM, glycolate oxidase, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been studied first by Lesk et al. in 1989. For these three proteins, there are twelve residues packed in an orderly way at the center of the barrel. These residues form three layers perpendicular to the axis of the barrel. The side chains forming these layers originate from alternating b strands. In the case of TIM and Rubisco, the middle layer is formed by residues from the odd-numbered b strands and the two other ones by residues from the even-numbered ones, whereas for glycolate oxidase, the case is exactly the opposite. This was interpreted as a common solution for increased stability, rather than as evidence for a common evolutionary origin. Although this was later shown not to be necessarily true (Raine et al. 1994), the problem of the evolutionary origin is still not considered having been solved (Janecek & Bateman 1996). Searching for clusters of identical or similar amino acids in different TIM barrel proteins (Selvaraj & Gromiha 1998) showed that there is a large number of physicochemically similar clusters located in the same type of secondary structure positions in different TIM barrel proteins. They often occupy equivalent positions in the barrel and contain some key residues that could be important for folding. Thus, it was suggested that the TIM barrel fold could be a result of such physicochemically similar clusters coming together during folding, rather than globally similar sequences folding in the same way. 24

2.1.3.2 Evidence for divergent evolution from a common ancestor

Already in 1985, it was suggested by Lonberg & Gilbert that TIM barrel proteins could have evolved from ancestors containing smaller ba units. This theory was based on the observation that exons and introns in the gene that encodes for pyruvate kinase are arranged such that the coding region is divided into pieces encoding for discrete secondary structure. Many of the TIM barrel proteins have a phosphate-binding site near the C- terminus of the domain. This was first noticed for the three TIM barrel enzymes (TrpF, indole-3-glycerol-phosphate synthase (TrpC), and the a -subunit of tryptophan synthase (TrpA)) along the tryptophan biosynthesis pathway (Wilmanns et al. 1991). A similar phosphate-binding site located near the loop connecting b strand 7 to a helix 7 and the N-terminal part of a helix 8 was also found in TIM, Rubisco, glycolate oxidase, flavocytochrome b2, trimethylamine dehydrogenase, and fructose-bisphosphate aldolase (Wilmanns et al. 1991), which are not functionally related to the enzymes of the tryptophan biosynthesis pathway. Later on, this phosphate-binding site was either found or predicted from several other TIM barrel enzymes as well (Rondeau et al. 1992, Fox & Karplus 1994, Janecek & Bateman 1996, Bork et al. 1995). This phosphate-binding site often concerns glycine residues located at the loop connecting b strand 7 to a helix 7 and a short additional helical structure in the loop after b strand 8. Glycoside hydrolases are the largest family of TIM barrel proteins. This family can be divided into subfamilies, the evolutionary relationships of many of which have been investigated. Juers et al. (1999) have suggested an evolutionary model for b-galactosidase and many other glycoside hydrolase family members. 17- barley-endoglucanases could be considered as the archetype of the common ancestor for many of these proteins. The evolutionary steps would have included extending the C-terminal loops, incorporation of new domains, and modification of the active-site cleft. Raine et al. showed in 1994 that the two types of layers of residues inside the b barrel (as in TIM and Rubisco versus glycolate oxidase) could also originate from a common ancestor after some very simple genetic alterations. The enolase family deviates quite a lot from the stereotypic TIM barrel in having a bbaa unit in the beginning. Even a large rearrangement like this could be a result of circular permutation of a common ancestor gene (Copley & Bork 2000). The same could be true for methylmalonyl-CoA mutase (Mancia & Evans 1998). Apart from the common phosphate-binding site, many other TIM barrel enzymes that use cofactors have corresponding metal-binding sites (Nagano et al. 2002). Recently, a phylogeny of the evolution of TIM barrel enzymes from the central metabolic pathways has been constructed (Copley & Bork 2000), indicating that indeed, most of these enzymes share a common evolutionary origin. Already decades ago, it was proposed that enzymes catalyzing subsequent steps in metabolism, and even just enzymes within similar biochemical pathways could be related by evolution (Horowitz 1945, 1965). Indeed, recent studies have shown that many of the TIM barrel enzymes within, for example, 25

Fig. 5. Proposed model for the evolution of TIM barrel enzymes from an ancestral half barrel by gene duplication, fusion of the barrel halves, second gene duplication event, and further divergent evolution as proposed by Lang et al. (2000).

tryptophan and histidine biosynthesis pathways have a common origin (Lang et al. 2000, Höcker et al. 2001b). As already suggested based on sequence comparisons (Fani et al. 1995), two TIM barrel enzymes catalyzing successive steps in histidine biosynthesis - N-[(5’-phosphoribosyl)formimino]-5- aminoimidazole-4-carboxamide ribonucleotide isomerase (HisA) and imidazole glycerol phosphate synthase (HisF) - are structurally homologous (Lang et al. 2000). Furthermore, these enzymes consist of two (ba )4 half barrels that are related by twofold symmetry, suggesting that the gene encoding a (ba )8 (TIM) 26 barrel would be a result of gene duplication of a gene encoding a half barrel followed by fusion and further divergent evolution (Fig. 5). Deduced from sequence similarities, it seems that three TIM barrel enzymes from the tryptophan biosynthesis pathway (TrpA, TrpC, and TrpF) are of the same evolutionary origin as HisA and HisF. On the other hand, the folding pathway of TrpA and TrpF is known to follow the so called ”6+2” mechanism, where the first six ba units are folded first, followed by the two last ones (Higgins et al. 1979, Eder & Kirschner 1992, Zitzewitz & Matthews 1999). This, together with the idea of half barrels, could be considered somewhat confusing. On the other hand, it may be that the evolutionary history and the folding pathway of the present TIM barrel proteins are independent of each other. The TIM barrel structure seems to be particularly well suited for divergent evolution of function, after the active-site scaffold has been constructed for one type of reaction (Gerlt & Babbitt 2001). Considering evidence from most recent experimental studies (Höcker et al. 2001a), as well as sequence comparisons (Copley & Bork 2000), it does seem likely that at least most of the TIM barrel enzymes have evolved from a common half-barrel ancestor. This gives interesting new insights into the evolutionary research on many other apparent single-domain protein families (Henn-Sax et al. 2001). Furthermore, it has to be questioned, whether even the half-barrel is the smallest ancestral unit.

2.1.3.3 TIM barrels in different genomes

TIM barrel proteins can be found in the genomes of all three major kingdoms of life - bacteria, archaea, and eukarya (Gerstein 1997, Wolf et al. 1999, Gerstein 2000), suggesting that the TIM barrel fold evolved before the divergence of these approximately two billion years ago (Doolittle et al. 1996). Most TIM barrel proteins are involved in basic metabolism, common to all species, which partly explains the abundance of this fold (Gerstein 1997, Wolf et al. 1999). Furthermore, TIM barrels are more abundant in bacteria and archaea than eukaryotes (Wolf et al. 1999). In yeast, of all proteins, the ones having the TIM barrel fold are expressed most, while in the whole yeast genome, the TIM barrel fold is the fifth most abundant fold (Gerstein 1997). It has been shown that proteins involved in energy production - like TIM barrel proteins often are - are highly abundant in the transcriptome (Jansen & Gerstein 2000).

2.1.4 Folding and stability of TIM barrel proteins

Some data are available on the stability and folding of TIM barrel proteins. TrpF and TrpA have been shown to follow a similar folding pathway where the first six ba units fold first and subsequently associate with the two last ba units 27

(Higgins et al. 1979, Eder & Kirschner 1992, Zitzewitz & Matthews 1999). In both cases, the N-terminal fragment is folded resembling the barrel structure with the two last strands missing, whereas the C-terminal fragment does not fold on itself. It has also been suggested that the building block for folding in TrpA is the bab unit (Zitzewitz & Matthews 1999, Tsai & Nussinov 2001) and the addition of these building blocks one after the other increases the structure and stability of the folded protein. Computational studies have indicated the importance of clusters of hydrophobic residues acting as folding initiation sites (Mirny & Shakhnovich 1999). On the other hand, such nucleation clusters might also be formed by other than hydrophobic interactions, such as hydrogen bonds or salt bridges (Kannan et al. 2001). HisA and HisF have internal twofold symmetry, and both halves can fold independently and associate to form a functional TIM barrel (Höcker et al. 2001a). This suggests that, unlike TrpA and TrpF, these enzymes would follow a ”4+4” folding pathway. One of the main features making the TIM barrel structure highly stable is the packing of side chains inside the barrel to form the contact layers discussed above. Another stabilizing feature, probably of even more importance, is the presence of hydrophobic side chain interactions between the amphipathic a helices and the central b strands (Vtyurin & Panov 1995). The residues involved in these interactions are mainly those with branched hydrophobic side chains, like valine, isoleucine, and leucine. In the contact layers inside the barrel, some of the residues can be polar, like lysine, arginine, and glutamine. These residues form the top or bottom layer and interact with solvent atoms or form the active site (Helin et al. 1995). An important aspect in the stability of enzymes is that they have to be stable, but also flexible enough, to perform catalysis. This is particularily true for enzymes from species living in extreme temperatures. In these enzymes, increased stability is achieved normally by additional hydrogen bonds, salt bridges, or complementary hydrophobic surfaces. One of the structural features that make the TIM barrel such a versatile framework for creating different proteins with highly variable enzyme activities is the separation of catalytic properties and stability. As the active site is located at the C-terminus, and the N-terminus is believed to be largely responsible for the stability of the protein, different active sites can be easily constructed without interfering with stability. On the other hand, as can be seen in many TIM barrel proteins from extremophiles, increased stability can be achieved by small changes that do not affect catalytic power. Also the ability to form quaternary structures is important for the stability. For example, Thermatoga maritima and Pyrococcus woesei TIMs form tetramers, whereas TIMs from most other organisms are dimers (Maes et al. 1999, Walden et al. 2001). TrpF in mesophiles is monomeric, but dimeric and highly stable in T. maritima (Sterner et al. 1996, Hennig et al. 1997). 28

2.1.5 Engineering the TIM barrel fold

A lot of interest has been put in engineering the TIM barrel fold; this involves both attemps to design new TIM barrel proteins as well as modifications of existing enzymes. These studies have especially focused on increasing stability, optimizing the catalytic properties, forming chimeras of different TIM barrel enzymes, or creating completely new enzyme activities. HisA and TrpF are TIM barrel enzymes along the histidine and tryptophan biosynthesis pathways, respectively, catalyzing similar Amadori rearrangements of aminoaldoses into the corresponding aminoketoses. Although these two enzymes hardly share any sequence identity, a directed evolution approach was successfully used to create an enzyme having both TrpF and HisA activities. Only a single amino acid exchange was necessary (Jürgens et al. 2000). TrpF and TrpC catalyze subsequent steps in the tryptophan biosynthesis pathway, the product of TrpF being the substrate of TrpC. An attempt to engineer TrpF activity into TrpC, using a combination of rational design and random screening, has been described (Altamirano et al. 2000). Recently, these results have been questioned and further experiments are needed (Altamirano et al. 2002). Rational design has been used to change the product specificity of Novamyl, an industrially relevant glycoside hydrolase from merely linear oligosaccharides to both linear and cyclic ones (Beier et al. 2000). Often, thermophilic enzymes have very low activity at mesophilic temperatures. This is thought to be a consequence of enhanced rigidity (Jaenicke 2000). The rate of a hyperthermophilic TrpC at 37°C could be improved by using random mutagenesis and in vivo selection (Merz et al. 2000). In this case, the rate- limiting step was product release which could be accelerated by making the loop structure involved more flexible. Recently, an artificial idealized TIM barrel protein was designed de novo (Offredi et al. 2003). The gene was constructed, and the resulting gene product could be expressed and purified as a stable, non-aggregated protein. Furthermore, this protein has a secondary-structure content similar to natural TIM barrels, appears to form quaternary structures, and behaves like a folded protein rather than a molten globule. Further studies to improve the solubility of this protein using in silico design and in vitro directed evolution are underway. Many efforts have also been undertaken to engineer new substrate specificity for TIM; these will be discussed in section 2.2.5.

2.2 Triosephosphate isomerase

Detailed studies on the structure and mechanism of TIM have been carried out for decades, especially by the groups of Jeremy Knowles and Gregory Petsko. Also, excellent reviews on the mechanism of this ”perfect enzyme” have been published (Knowles & Albery 1977, Knowles 1991). The studies reported in this thesis have been performed using chicken, Trypanosoma brucei brucei, and 29

Leishmania mexicana mexicana TIM. Except for the human disease-related mutations, the numbering scheme used in this thesis refers to that of the trypanosomal TIMs. For example, the catalytic triad in these organisms is Lys13, His95, and Glu167.

2.2.1 Structural features

2.2.1.1 The overall structure

To date, the amino acid sequence of approximately 60 TIMs from different species is known. TIMs from different organisms show on average 50 % pairwise sequence identity. The trypanosomal TIMs have many positively charged residues on the surface leading to a high pI compared to TIMs from other species, which are neutral (Misset et al. 1986). The b strands of TIM are rather short (3-6 residues). The a helices are significantly longer (6-20 residues). The N- terminal loops that connect the a helices to the next b strands are short, whereas the C-terminal loops that connect the b strands to the following a helices are longer and play an important role in substrate binding and catalysis. Most of the fully conserved residues are near the active-site pocket, and thus, they are most likely important for the architecture of the active site. The only exception is a salt bridge between Arg191 and Asp227 or 229. This salt bridge is 20 Å away from both the active site and the dimer interface, and links together the C-terminal ends of helices 6 and 7 (Wierenga et al. 1992). This anchoring interaction has been proposed to be important for the functional flexibility of loops 6 and 7 at the other end of these helices. The eight b strands and a helices form a classical TIM barrel fold, in which the strands and helices are connected by loops of variable length (Fig. 1). Two of the loops protrude from the globular structure - namely the dimer interface loop 3 and the substrate binding loop 6. Most TIMs are homodimers which form as one of the C-terminal loops (loop 3) gets buried inside the adjacent subunit during folding. Two exceptions are known from the thermophilic bacteria T. maritima and P. woesei, which have tetrameric TIMs (Maes et al. 1999, Walden et al. 2001).

2.2.1.2 Folding and stability

All known wild-type TIMs form oligomers, and the catalytic activity of the engineered monomeric variants is severely impaired. Still, the reason for TIM to form at least dimers is unknown, as no allostery between the subunits exists. It has been proposed that the reason would be either the gain in stability upon subunit assembly (Mainfroid et al. 1996b) or that dimerization is necessary for the completion of the active-site pocket (Schliebs et al. 1997). 30

Maes et al. (1999) have performed a comparative analysis on the thermostability properties of ten different TIM structures including two thermophilic (T. maritima and Bacillus stearothermophilus) and one psychrophilic (Vibrio marinus) enzyme. Of these, T. maritima TIM is a tetramer. It was found that both in thermophilic and psychrophilic TIMs, an increased amount of salt bridges is present. In the thermophilic enzymes, more hydrophobic surface was seen to be buried during folding compared to other TIMs. In the dimer interface of L. mexicana TIM, a Gln present in all other TIMs (Williams et al. 1999) is replaced by a Glu. This is thought to be a destabilizing feature, but still, the L. mexicana TIM is as stable as the other ones. Thus, other interactions must be present to compensate for the introduction of an ionizable group in the buried interface. Mutating this Glu to Gln (the E65Q mutant) results in a superstable enzyme with catalytic properties similar to those of the wild- type enzyme (Williams et al. 1999). The Tm of this variant is almost 30°C higher than for the wild type.

2.2.1.3 The dimer interface

The dimer interface is formed mainly by loops 1-4. Loop 3 protrudes from the molecule and extends into a deep pocket formed by loops 1, 2, and 4 of the adjacent subunit interacting with Lys13 and Glu97 via van der Waals contacts and hydrogen bonds. For example, a hydrogen bond is formed between Thr75 in loop 3 and the carboxylate group of Glu97 (Schliebs et al. 1996). As two of the active-site residues come from loops 1 and 4, the active site actually is at the dimer interface, although all the residues directly interacting with the substrate come from the same subunit. The interactions between loop 3 and loops 1 and 4 from different subunits influence the exact positioning of active-site residues Lys13 and His95 from loops 1 and 4, respectively.

2.2.1.4 Active-site construction

The active site of TIM, like that of all TIM barrel proteins, is located at the C- terminal end of the b barrel (Fig. 6). The substrate - a triose sugar - is rather small, and the phosphate moiety just fits in a very tight binding pocket lined by residues from loops 6, 7, and 8. Consequently, TIM has a very narrow substrate specificity, the only known substrates being DHAP and DGAP. Loops 6 and 7 go through a conformational change as the ligand is bound (Alber et al. 1981, Lolis & Petsko 1990, Noble et al. 1991a,b, Verlinde et al. 1991, Wierenga et al. 1991b). In the unliganded state, loop 6 interacts with loop 5, whereas in the liganded state, it interacts with the YGGS motif (residues 210-213) of loop 7. Oh of Tyr210 and Og of Ser213 are hydrogen bonded to the main chain nitrogen atoms of Ala178 and Gly175 in loop 6, respectively. N of Gly173 is hydrogen bonded to one of the 31 phosphate oxygens of the substrate. In the conformational switch from the open to the closed state, the tip of loop 6 (near Gly175) moves 7 Å as a rigid body (Wierenga et al. 1991b) (Fig. 7), whereas in loop 7, the peptide planes after Gly211 and Gly212 rotate 90° and 180°, respectively. The closure of loop 6 has also been studied by nuclear magnetic resonance (NMR) (Williams & McDermott 1995, Rozovsky & McDermott 2001, Rozovsky et al. 2001) and molecular dynamics calculations (Joseph et al. 1990, Derreumaux et al. 1998). The calculations indicate that the loop starts to open in the region Lys176-Val177 (Joseph et al. 1990), and that this is initiated by the loosening of the hydrogen bond between Gly175 and Ser213 (Derreumaux et al. 1998). The opening/closing motion of loops 6 and 7 appears to be partially rate limiting for the catalysis of the reaction in both directions (Rozovsky & McDermott 2001, Rozovsky et al. 2001). It is unclear which structural features are associated with the energy barrier of this conformational switch (Derreumaux et al. 1998, Noble et al. 1993, Yüksel et al. 1994).

E167

H95

K13

Fig. 6. The active site of TIM (5TIM; Wierenga et al. 1991a) at the C-terminus of the b barrel. The catalytic residues Glu167, His95, and Lys13 are shown. The figure was generated with Molscript (Kraulis 1991). 32

Fig. 7. The open and closed conformations of loop 6 superimposed. Shown in light gray is the closed form of the loop with PGH bound in the active site and the Glu167 sidechain in the swung-in conformation (1TPH; Zhang et al. 1993) Shown in dark gray is the open form with the Glu167 side chain in the swung-out conformation (5TIM; Wierenga et al. 1991a). The figure was generated with DINO (Philippsen 2000)

Loop 6 can be divided into an N-terminal hinge (Pro168, Val169, and Trp170), a rigid tip (Ala171, Ile172, Gly173, Thr174, and Gly175), and a C-terminal hinge (Lys176, Val177, and Ala178) (Sun & Sampson 1998, 1999). After this, helix 6 starts with Thr179 as the N-cap residue, followed by Pro180 in the N-cap+1 position. The tip of the loop moves as a rigid body, while small changes in the f and y angles of the N- and C-terminal hinge cause loop opening and closure. The largest changes occur for y (Lys176) and f (Val177), which are +57 and -38°, respectively (Wierenga et al. 1991b). Solid-state NMR studies have shown that the opening of loop 6 is not ligand-gated; instead, also in the liganded state, the loop opens at a rate in the millisecond timescale (Williams & McDermott 1995). The group of Nicole Sampson has used random mutagenesis to create new functional amino-acid triplets to the C-terminal (Sun & Sampson 1998) and N- terminal (Xiang et al. 2001) hinge regions of loop 6 and studied the kinetic properties these mutants. It can be seen that significant sequence variability is 33 allowed for the C-terminal region. Much less variability is possible for the N- terminal region without interfering with the catalytic activity. Ser96 is a conserved residue in most TIM sequences. An exception is TIM from Plasmodium falciparum, which has a phenylalanine in this position (Parthasarathy et al. 2002a,b). In the open conformation of loop 6, the catalytic glutamate side chain is hydrogen bonded to the main chain and side chain of Ser96. Mutating this serine to proline decreases the catalytic activity compared to the wild-type enzyme, but in the H95N and E167D mutants, it is able to recover some of the lost activity (Komives et al. 1995, 1996). The catalytic residues Glu167 (165 in e.g. chicken TIM), His95, and Lys13 come from loops 6, 4, and 1, respectively (Knowles 1991). Residues from each of the b strands (or subsequent loops) contribute to the active site. Thr75 from loop 3 of the opposing monomer is hydrogen bonded to the side chains of Asn11 and Glu97, thus providing essential stabilizing interactions. Glu167 in the beginning of loop 6 displays two distinct conformations - the ”swung-out” conformation in the unliganded enzyme and the ”swung-in” conformation in the liganded form (Davenport et al. 1991, Lolis et al. 1990, Noble et al. 1991a, 1993). In the unliganded enzyme, the carboxylate group of the glutamate is hydrogen bonded to Ser96. Ligand binding induces a conformational switch, where the glutamate side chain moves by 2-3 Å getting buried in the protein. During this process, the hydrogen bonds to Ser96 are broken and replaced by contacts to the ligand (Lolis & Petsko 1990, Noble et al. 1993). Lys13 is present in a somewhat strained conformation, as it is an outlier in the Ramachandran plot (Ramachandran et al. 1963) of all TIM structures, with positive f and negative j values (Noble et al. 1993). Evidently, the exact positioning of this side chain to create a positively charged environment for the negatively charged intermediate to bind is important for the catalytic mechanism. Other conserved residues near the active site are Asn11, Glu97, Ile172, Leu232, and Cys126. The role of His95 is not fully understood: it may take part in shuttling protons during the reaction cycle, or it may be present just to stabilize the reaction intermediate, or both (Davenport et al. 1991, Komives et al. 1991). From the structure, it can be deduced that Nd is unprotonated as it is hydrogen bonded to the peptide NH of Glu97. If the histidine acts as a neutral imidazole (protonated on Ne and unprotonated on Nd), it would stabilize the transition state by hydrogen bonding. The neutral imidazole could also act as a catalytic acid to protonate the substrate. This would lead to a fully unprotonated, negatively charged, histidine side chain, as has been postulated based on NMR (Harris et al. 1997) and theoretical (Åqvist & Fothergill 1996) studies. 34

2.2.2 The gene, expression, and cellular localization of triosephosphate isomerase

In the human genome, there is only one functional gene encoding TIM. This is located in chromosome 12 (at 12p13) together with the genes for glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase B, and enolase 2 (Rethore et al. 1977, Law & Kao 1979). Three TIM pseudogenes from different chromosomes have been identified that evidently have diverged approximately 18 million years ago (Brown et al. 1985). The functional gene spans 3.5 kb and is split into 7 exons. TIM is highly expressed in virtually all cells. In most organisms, TIM is localized in the cytosol where glycolysis happens. Trypanosomal organisms have special peroxisome-like organelles, glycosomes, in which glycolysis takes place. In these species, for example T. brucei and L. mexicana, TIM is found both in the cytosol and glycosomes (Coombs et al. 1982, Hart & Opperdoes 1984, Mottram & Coombs 1985). Still, only a single gene encoding TIM has been found from L. mexicana and T. brucei (Michels 1986), implying that a certain fraction of the protein is transported into the glycosomes (Kohl et al. 1994). The glycosomal targeting signal in TIM has not been identified, but the fact that the trypanosomal TIMs are highly basic with pIs of nearly 10 compared to 5.4 and 6.5 of the yeast and chicken enzymes, respectively (Misset et al. 1986), has been considered as a possible recognition feature (Wierenga et al. 1987).

2.2.3 The function of triosephosphate isomerase

2.2.3.1 Glycolysis

Glycolysis is the metabolic pathway that is used in almost all living cells to degrade carbohydrates in order to produce energy and carbon compounds to be used in biosynthetic pathways. Glycolysis is an anaerobic process that takes place in the cytosol, and probably evolved very early in the history of life - before the development of eukaryotic species. In aerobic organisms, glycolysis is followed by the citric acid cycle and the electron transport chain. Glycolysis consists of ten distinct reactions, for which ten different enzymes are required (Fig. 8). The net gain of glycolysis is two molecules of adenosine 5’-triphosphate (ATP) and two molecules of pyruvate from one molecule of glucose. In the first stage, glucose is converted to fructose 1,6-bisphosphate via two phosphorylation reactions and one isomerization. Hexokinase (Mulichak et al. 1998, Rosano et al. 1999, Aleshin et al. 2000) catalyzes the phosphorylation of glucose at the oxygen attached to the C6 carbon of glucose to form glucose-6- phosphate. Phosphoglucose isomerase (Sun et al. 1999, Chou et al. 2000, Jeffery et al. 2000, Davies & Muirhead 2002) catalyzes the interconversion between 35

CHO CHO CH2OH H C OH H C OH Phosphoglucose C O HO isomerase C H Hexokinase HO C H HO C H H C OH H C OH H C OH H C OH H C OH H C OH ATP ADP 2- 2- CH2OH CH2OPO3 CH2OPO3 Glucose Glucose-6-phosphate Fructose-6-phosphate ATP CH2OH Phosphofructo- kinase C O ADP 2- CH2OPO3 CH2OPO3 2- DHAP C O HO C H TIM Aldolase H C OH CHO H C OH 2- H C OH CH2OPO3 2- Fructose-1,6-bisphosphate CH2OPO3 DGAP Pi NAD+ GAP de- hydrogenase NADH 2- COOPO3 Phosphoglycerate kinase COO - H C OH H C OH 2- 2- CH2OPO3 CH2OPO3 1,3-bisphosphoglycerate ADP ATP 3-phosphoglycerate

Phospho- glycerate mutase - - COO - COO COO Enolase Pyruvate kinase 2- 2- C O C OPO3 H C OPO3 CH3 CH2 CH2OH ATP ADP Pyruvate Phosphoenolpyruvate 2-phosphoglycerate

Fig. 8. The reactions and enzymes involved in glycolysis. The amounts of ATP and NADH produced are indicated per one molecule of DGAP, rather than glucose. 36 glucose-6-phosphate and fructose-6-phosphate. Fructose-6-phosphate is then phosphorylated once more to form fructose-1,6-bisphosphate by phosphofructokinase (Evans et al. 1981, Shirakihara & Evans 1988, Schirmer & Evans 1990). Two molecules of ATP are consumed in the phosphorylation reactions. In the second stage, fructose-1,6-bisphosphate is cleaved to DHAP and DGAP by aldolase (Blom & Sygusch 1997, Choi et al. 1999, Dalby et al. 1999, Hall et al. 1999). DHAP is used in the biosynthesis of lipids, but DGAP continues into the glycolytic pathway. TIM catalyzes the interconversion of these two metabolites. DGAP is oxidized to 3-phosphoglycerate. This is the first ATP-producing step in glycolysis, Two distinct reactions catalyzed by two enzymes are required. The first step produces glycerate-1,3-bisphosphate and is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (Mercer et al. 1976, Yun et al. 2000). The second step leading to 3-phosphoglycerate is catalyzed by 3- phosphoglycerate kinase (Bernstein et al. 1997, Auerbach et al. 1997, McPhillips et al. 1996). Since two molecules of DGAP are formed from each molecule of glucose, this step produces two molecules of ATP per molecule of glucose. In the last stage, 3-phosphoglycerate is converted to pyruvate via three reactions. Again, two molecules of ATP are formed for each molecule of glucose. Phosphoglyceromutase (Rigden et al. 1999, Crowhurst et al. 1999) catalyses the interconversion between 3-phosphoglycerate and 2-phosphoglycerate. The next interconversion between 2-phosphoglycerate and phosphoenolpyruvate is catalyzed by enolase (Lebioda & Stec 1991, Lebioda et al. 1991, Zhang et al. 1994b). Finally, phosphoenolpyruvate is converted to pyruvate by pyruvate kinase (Jurica et al. 1998, Larsen et al. 1998), producing ATP. One of the goals of the structural genomics (Burley et al. 1999) efforts is to solve the three-dimensional structures of all enzymes related to biochemical pathways, and eventually, to understand the detailed catalytic mechanisms of these. One of the first solved and probably best characterized pathways is the glycolytic pathway. The structures of all the ten enzymes along this pathway are known (reviewed by Erlandsen et al. 2000). Four of the glycolytic enzymes have adapted the TIM barrel fold, namely TIM, aldolase (Blom & Sygusch 1997, Choi et al. 1999, Dalby et al. 1999, Hall et al. 1999), enolase (Lebioda & Stec 1991, Lebioda et al. 1989, 1991, Zhang et al. 1994b), and pyruvate kinase (Jurica et al. 1998, Larsen et al. 1998).

2.2.3.2 The catalytic mechanism

TIM catalyzes the interconversion of DHAP and DGAP (Fig. 9) during glycolysis. The catalytic mechanism has been studied by the means of X-ray crystallography, NMR, spectroscopy, mutagenesis, and enzymology, including the use of labeled substrates. In addition, several computational approaches have been used to complement the experimental results. The mechanism concerns proton transfers via enediol(ate) intermediates, which is also believed 37 to be the case for e.g. phosphoglucose isomerase (Davies & Muirhead 2002). No cofactors are needed, and TIM has been referred to as the ”perfect enzyme”, its rate being limited only by the diffusion of the substrate and product on and off the enzyme surface (Knowles & Albery 1977, Knowles 1991). Some other aldose- ketose isomerases have adopted a different strategy for the same reaction. For example xylose/xylulose isomerase is a metal-dependent enzyme utilizing Mg2+ to assist in the hydride shift and the preceeding and following proton transfer steps (Asboth & Naray-Szabo 2000, Garcia-Viloca et al. 2003). The kcat of xylose isomerase is approximately 1000-fold lower than that of TIM (Lambeir et al. 1992). This enzyme has been thoroughly studied, as it is important for biotechnological industry in the conversion of glucose to fructose.

H H O H H O H C H O P O C C OH O P O C C C O O H O O H OH

DHAP DGAP

Fig. 9. The reaction catalyzed by TIM is the reversible isomerization between a ketone (DHAP) and an aldehyde (DGAP).

Although the basics of the TIM reaction are fairly well understood, the exact chemical details of the mechanism are still unclear. There is general agreement about the mechanism of the first and last chemical steps in the reaction. The reaction is started with the abstraction of the pro-R proton from the C1 carbon of the substrate (DHAP) by Glu167, leading to a planar enediolate intermediate. In the absence of enzyme, this is energetically a very unfavourable reaction, due to the high pKa of the C1 atom of the substrate. For example, the pKa of methane is estimated to be 40 (Cleland 1998). Therefore, proton removal from a carbon atom is very difficult. If the resulting carbanion is stabilized, for example in the presence of an adjacent carbonyl function, then the pKa is much lower, near 20 (Zhang et al. 1999). This is still considerably different from the pKa of a glutamate side chain which is known to extract the proton from DHAP in the first step of the TIM reaction (Knowles 1991, Knowles & Albery 1977). In the enzyme environment, several residues contribute to facilitating this step by stabilizing the developing negative charge. The forming enediolate intermediate is stabilized via strong hydrogen bonds. Such bonds, if unusually short and strong, are called low-barrier hydrogen bonds (LBHB). The possible role of LBHBs in the mechanism of TIM, as well as other enzymes, has been discussed since 1993 (Gerlt & Gassman 1993, Cleland & Kreevoy 1994, Cleland et al. 1998). NMR studies have suggested that in the complex of TIM with the transition-state 38 analogue phosphoglycolohydroxamate (PGH), there exists an LBHB between the N-OH hydrogen and the catalytic glutamate (Harris et al. 1997). Lodi and Knowles (1991) suggest that the resulting enediolate is stabilized by a strong hydrogen bond to the neutral His95. Three alternative pathways have been suggested for the intermediate steps following the initial proton abstraction (reviewed in Cui & Karplus 2001) (Fig. 10). In the ”classical” mechanism (referred to as path A in Fig. 10), the proton abstracted by Glu167 is delivered directly to the adjacent carbon, unless, in the mean time, exchanged with solvent (Knowles & Albery 1977). A requirement for this mechanism is that the proton transfer between the oxygens is mediated by the His95 side chain (Bash et al. 1991, Lodi & Knowles 1991). In path B, this proton is transfered straight to O2 of the substrate without any direct contribution from the histidine side chain (Alagona et al. 1984, 1986, 1995). Recent computational studies suggest that this path is unlikely to be significant (Peräkylä & Pakkanen 1996, Cui & Karplus 2002). In the ”criss-cross” mechanism (path C), the glutamate is the only catalytic residue (Harris et al. 1997, 1998, Nickbarg et al. 1988, Pompliano et al. 1990, Zhang et al. 1999, Peräkylä 1997, Peräkylä & Pakkanen 1996). This mechanism requires several movements of the glutamate side chain during catalysis. NMR experiments have shown that in wild type TIM, both the ”classical” (path A) and the ”criss-cross” (path C) mechanism contribute to the reaction (Harris et al. 1997, 1998). The ”criss-cross” mechanism has been proposed to function in the H95Q mutant, which displays a 200-fold decrease in the catalytic rate (Nickbarg et al. 1988, Komives et al. 1991). Some of the activity of this mutant can be recovered by mutating Ser96 to Pro. This is expected to facilitate loop closure and the conformational switch of the catalytic glutamate from the swung-out to the swung-in position (Komives et al. 1991). The last step from the enediolate to DGAP is very similar to the first one, but this is an exothermic process since an acidic proton is transferred from the glutamate to the C2 atom of the enediolate intermediate to form the product. In the TIM reaction, an important consideration is thought to be that, in order to avoid phosphate elimination, the bridging oxygen of the phosphate group of the substrate has to be held in plane with respect to the ketone oxygen of the triose moiety, such that electrons cannot be delocalized (Lolis & Petsko 1990), analogous to, for example, the decarboxylation reaction in the case of malic enzyme (Cleland 1998). This is achieved with conserved interactions within the very tight phosphate-binding pocket. In the absence of loop 6, the reaction intermediate is lost into solution, and degraded into inorganic phosphate (Pi) and methylglyoxal, which is toxic to the cell (Pompliano et al. 1990). The same is observed for some mutants, in which the catalytic turnover is very slow (Komives et al. 1996). Excluding solvent from the active site by the closure of loop 6 is also expected to enhance the basicity of the catalytic glutamate (increasing its pKa), thus facilitating its role in abstracting the proton from the carbon of the substrate. In the closed conformation, the Glu167 side chain is not hydrogen bonded to any of the protein atoms, but, in addition to the substrate, only to a water molecule that is part of a water network which extends to the bulk solvent. The 39

Glu167 Glu167

O O HO O

H O H H O O P O OH O P O - OH O O O O

DHAP HN HN N N His95 His95

A B C

Glu167 Glu167 Glu167

HO O HO O O O

H H H O O O O P O OH O P O - O O P O OH O OH O OH O OH

N HN HN - N N N His95 His95 His95

Glu167

O O

H O H O P O O O HO HN DGAP N His95

Fig. 10. Alternative pathways for the reaction catalyzed by TIM. The reaction is initiated by the abstraction of the pro-R proton of C1 of DHAP by Glu167, as indicated by the arrows. Path A is also referred to as the classical mechanism and path C as the criss-cross mechanism.

importance of these waters for catalysis has been discussed by Zhang et al. (1999). The presence of some water molecules in the otherwise rather hydrophobic active site is expected to have an effect on the pKa of the glutamate 40 side chain as well as on the binding of the substrate or intermediates. Moreover, the position of the glutamate side chain may be influenced by the ordered water molecules nearby. Proton exchange between the catalytic glutamate and bulk solvent via this network might also explain the low yield of tritium transfer from the labeled C1 of DHAP to C2 of DGAP during catalytic turnover (Knowles & Albery 1977).

2.2.3.3 Substrate and transition-state analogues

Three planar transition-state analogues, 2-phosphoglycolate (PGA), PGH, and 2- (N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) (Fig. 11), have been most succesfully used for studying the structural details of the reaction mechanism of TIM (see, for example, Lolis & Petsko 1990, Davenport et al. 1991, Noble 1992). Other ligands used in structural studies include at least 3-phosphonopropionate (Noble et al. 1991a), 2-phosphoglycerate (Noble et al. 1991b), 3-phosphoglycerate (Noble et al. 1991a), and glycerol-3-phosphate (Parthasarathy et al. 2002a).

H O H H H O H N OH O P C C N C O P O C C O H H OH O O H O

PGH IPP

H

O H O11

O P O C2 C 1

O H O12

PGA

Fig. 11. Three planar transition-state analogues of the TIM reaction. PGH and PGA are analogues of the ketone substrate, DHAP, and IPP is an analogue of the aldehyde substrate, DGAP. The numbering scheme for PGA is shown.

The mode of binding of PGA to TIM has been studied ever since this compound was proposed to be a transition-state analogue of the TIM reaction 41

(Wolfenden 1969). 31P-NMR studies indicate that PGA binds to TIM as a trianion (Campbell et al. 1979). It is assumed that the tight binding of PGA is largely due to electrostatic interactions between PGA and the protonated Glu167 and neutral His95 side chains (Lolis & Petsko 1990). PGA is a true transition- state analogue as it has the same planarity and charge as the intermediate formed from the substrate after proton abstraction by the catalytic glutamate. The true substrate differs from PGA such that the O11(H) group is the C1(H)- OH moiety in the ketone substrate (DHAP). PGA is a transition-state analogue in which the hydroxymethyl group of DHAP is replaced by a hydroxyl group. The O12 atom mimics the ketone moiety, whereas the O11 atom mimics the C1 atom of DHAP from which Glu167 abstracts a proton. PGH is a transition-state analogue mimicking structural features of the ketone substrate, DHAP (Collins 1974). The TIM-PGH complex is thought to very well describe the true intermediate of the reaction, as the oxygens in the planar hydroxamate moiety of PGH are thought to occupy similar positions as the

Table 2. Kinetic constants of TIM from several species. All other measurements have been performed at 25°C, except for the psychrophilic V. marinus values, which were measured at 10°C. ______Species Km(mM) kcat (min-1) Reference DGAP DHAP DGAP DHAP ______

Human 0.49 2.7x105 Mande et al. 1994 Lu et al. 1984

Chicken 0.47 0.97 2.56x105 2.59x104 Putman et al. 1972 0.39 1.57 2.60x105 2.92x104 Plaut & Knowles 1972

Rabbit 0.39 6.3x105 Lambeir et al. 1987 0.32 1.23 5.1x105 5.2x104 Krietsch et al. 1970 0.32-0.46 0.62-0.87 Hartman & Norton 1974

Yeast 1.22 7.9x105 Lambeir et al. 1987 1.45 Hartman & Ratrie 1977 1.27 1.23 1.0x106 4.9x104 Krietsch et al. 1970

T. brucei 0.25 1.2 3.7x105 6.5x104 Lambeir et al. 1987

L. mexicana 0.31 1.1 2.4x105 2.2x104 Kohl et al. 1994

V. marinus 1.9 4.2x105 Alvarez et al. 1998

E. coli 1.03 5.4x105 Alvarez et al. 1998 ______42 oxygens of the planar enediolate moiety of the transition state (Davenport et al. 1991, Zhang et al. 1994a, Komives et al. 1996). IPP was originally synthesized for trypanosomal drug design purposes (Noble 1992, Witmans 1995). It is a phosphonate compound, as the bridging oxygen is replaced with a carbon. Its structure resembles PGH, but it is an analogue of DGAP, rather than DHAP.

2.2.3.4 Kinetic properties

TIM is a very efficient enzyme - it can speed up the isomerization reaction by a factor of 109, and the reaction rate is limited by the rate of diffusion of the substrate and product on and off the enzyme surface (Blacklow et al. 1988). No cofactors are needed for the reaction, nor has any allosteric cooperation between the two subunits been noted. The physiologically more relevant reaction is the consumption of DHAP and formation of DGAP, which is needed in glycolysis. The equilibrium strongly favours the conversion of DGAP into DHAP, whith a Keq of 420 (Reynolds et al. 1971). Even in the presence of enzyme, if DGAP is not consumed, the formation of DHAP is the predominant reaction. The rate- limiting step for the formation of DHAP is diffusion and for the formation of DGAP the consumption of DGAP in glycolysis (Knowles et al. 1972). The kinetic constants of TIM from several species, as reported in the literature, are summarized in Table 2. In addition to the substrate or transition-state analogues

Table 3. Inhibition constants for several inhibitors of TIM listed for T. brucei TIM. ______Inhibitor Ki (mM) Reference ______

PGA 0.027 Lambeir et al. 1987

PGH 0.008 Schliebs et al. 1996

Glycerol-1-phosphate 0.61 Lambeir et al. 1987

3-phosphoglycerate 1.3 Lambeir et al. 1987

2-phosphoglycerate 6.9 Lambeir et al. 1987

3-phosphonopropionate 27.0 Noble et al. 1991a sulphate 4.5 Lambeir et al. 1987

Phosphate 4.5 Lambeir et al. 1987

Arsenate 4.6 Lambeir et al. 1987 ______43 discussed in section 2.2.3.3., sulphate, phosphate, and arsenate act as inhibitors binding to the phosphate-binding pocket in the active site. Inhibition constants for the most common inhibitors are shown in Table 3.

2.2.4 Triosephosphate isomerase deficiency

Several mutations causing severe symptoms of TIM deficiency have been characterized (for review, see Schneider 2000). Patients suffering from TIM deficiency display severe haemolytic anemia and often progressive neuromuscular degeneration. Often, higher rates of infections are also observed. TIM deficiency is a rare autosomal recessive disorder, and most of the patients die at very early age. Several mutations are known to occur in the upstream regulatory region of the gene encoding TIM. In addition, there is a mutation in which the starting methionine codon ATG is mutated to AAG, and several frame-shift mutations have been reported (Schneider 2000). As these mutations result in the lack of TIM, they will not be discussed here in more detail. Instead, several amino acid substitutions that alter either the catalytic mechanism, stability, or folding of TIM are described in this section. All known disease- related mutations in the TIM sequence are listed in Table 4. The most common mutation causing TIM deficiency is Glu104Asp. This mutation leads to a thermolabile enzyme (Daar et al. 1986). All other mutations have been found in single cases only. The Glu104Asp mutation most likely origins from a single mutation in a common European ancestor (Arya et al. 1997). Heterozygotes carrying this mutation are clinically normal, but in some cases, compound heterozygosity with some other mutation in the other allele is observed to cause TIM deficiency (Arya et al. 1997). In two cases, this mutation is Cys41Tyr and in one case, Ile170Val. In one case, Arg189 codon is mutated to a stop codon resulting in a truncated enzyme. Glu104 is strictly conserved in TIM sequences, and the molecular basis for the disease caused by this mutation can also be deduced from the crystal structures available. As seen, for example, in the human TIM structure (Mande et al. 1994), this residue is almost completely buried in the dimer interface of the two subunits. The sidechain of Glu104 lies between two positively charged residues; Arg98 and Lys112. Arg98 is also forming a salt bridge to Glu77 and is probably needed for positioning the catalytic His95 to a position optimal for catalysis. Mutating Glu104 to Asp shortens the side chain by one carbon atom, leading to an unfavourable, destabilizing interaction of Arg98 and Lys112 side chains and possibly dispositioning of His95 in the active site (Daar et al. 1986, Mande et al. 1994). One more mutation described in one patient is Phe240Leu (Chang et al. 1993). This mutation also causes decreased thermostability of the enzyme. The molecular basis of this mutation can be explained from the structure as well (Mande et al. 1994). Phe240 is located in loop 8, such that it contacts b strand 1 and the phosphate-binding helix. Thus, mutating this residue may affect both substrate binding and stability, as b strand 1 is close to the dimer interface as 44

Table 4. Disease-related TIM mutations found in humans. ______Mutation Comments Reference ______-5A -> G, -8G -> A Upstream mutation Watanabe et al. 1996

-5A -> G, -8G -> A, Upstream mutation Watanabe et al. 1996 -24T -> G

MetInitLys Initiation codon Schneider & Cohen-Solal 1996

Leu28frameshift Frameshift Schneider & Cohen-Solal 1996

Cys41Tyr Dimer interface, loop 2 Arya et al. 1995

Gly72Ala Dimer interface, loop 3 Watanabe et al. 1996

Glu104Asp Dimer interface, loop 4, (most common mutation) Daar et al. 1986

Gly122Arg Backside of the molecule Perry & Mohren- weiser 1992

Glu145Stop Truncated enzyme Hollàn et al. 1997

Val154Met Backside of the molecule Watanabe et al. 1996

Ile170Val Active site, loop 6 Arya et al. 1995

Arg189Stop Truncated enzyme Daar & Maquat 1988

Val231Met b strand 8 Bardosi et al. 1990

Phe240Leu Loop 8 Chang et al. 1993 ______well as the active-site residues. Other mutations found in individual patients are Gly122Arg, Gly72Ala, Val154Met, Val231Met, and Glu145stop. When looking at all known disease- related mutations, they are located in three functional areas in the protein involved in substrate-binding, flexible loop 6, or dimer interface (Halfman & Schneider 1997, Arya et al. 1996). Most of the mutations occur in residues that are highly conserved among species, and the molecular basis of many of them can be understood from the three-dimensional structure. Surprisingly, the symptoms in TIM deficiency have very little to do with glycolysis, but they rather affect the neuromuscular system. The only predicted effect that TIM deficiency seems to have is the accumulation of DHAP in cells. It has been suggested that compensation for the less efficient glucose utilization 45 and especially ATP production could occur via the pentose phosphate pathway (Schneider 2000).

2.2.5 Protein engineering studies on triosephosphate isomerase

A monomeric variant of T. brucei TIM has been designed by shortening the dimer-interface loop 3 (Borchert et al. 1994). Fifteen mostly hydrophobic residues of this loop have been replaced by 8 more hydrophilic ones. The monomeric variant - called MonoTIM - is soluble and stable, but displays only one thousandth of the activity of the dimeric wild type enzyme (Schliebs et al. 1996). The crystal structure of MonoTIM shows that the positions of loops 1 and 4 are severely affected by the shortening of loop 3. Loop 1 with the catalytic lysine is disordered to the extent that it is invisible in the electron density maps. In another experiment, two point mutations have been introduced to the tip of loop 3 to form a monomer, but the activity of this variant was as low as that of the original MonoTIM (Schliebs et al. 1997). Monomeric variants of human TIM, carrying point mutations, have also been characterized (Mainfroid et al. 1996a). Combining modeling with mutagenesis and crystallography, variants of MonoTIM have been designed, where the phosphate-binding loop 8 has been shortened, in order to make the active-site pocket wider and capable of binding substrates other than DHAP and DGAP (Norledge et al. 2001). One of the resulting proteins - ml8bTIM - is stable but inactive. The crystal structure shows that the new loop 8 is in a well-defined conformation, and the binding pocket has indeed been widened. The flexibility of loops 6 and 7, as well as the correct positioning of the catalytic residues, seem to have been preserved in ml8bTIM. Saab-Rincón et al. (2001) have used the directed evolution approach to improve the catalytic activity of MonoTIM. It seems that the loss of activity in the monomeric variants is largely due to increased flexibility of the active-site loops, as the stabilizing interactions with the other subunit have been lost. Replacing these intersubunit interactions with intrasubunit ones might lead to recovery of activity. The strategies followed included randomizing the whole gene sequence as well as randomizing only loop 2. Interestingly, both approaches resulted in two mutations (A43P and T44A/S), which show a 11-fold increase in kcat. 3 Aims of the present study

Although TIM has been extensively studied over more than 30 years, the structure-function relationships of this enzyme, like many others, are far from being fully understood. The initial questions at the beginning of this study mainly concerned:

- the importance of the fully conserved salt bridge between Arg191 and Asp227 far from both the active site as well as the dimer interface (III)

- properties of the hinge regions in the flexible substrate-binding loop 6.

The latter studies are being done in collaboration with the group of professor Nicole Sampson at the State University of New York. This work is still in progress and will be described elsewhere. In quite early stages of the study, a side project, concerning the superstable E65Q mutant of L. mexicana TIM, was initiated. During this time, also crystals diffracting to atomic resolution were obtained. This has lead the focus of this study to:

- solving the structure of the E65Q mutant complexed with PGA (I)

- determining the binding mode of an analogue of the aldehyde substrate, IPP (II)

- studying the exact chemical details of the catalytic cycle, such as the flexibility of the substrate and its binding mode at atomic resolution (IV). 4 Materials and methods

Detailed descriptions of most methods can be found in the original articles referred to by their Roman numerals.

4.1 Recombinant protein expression and purification (I-IV)

The T. brucei and L. mexicana TIM proteins and all the mutants were expressed in E. coli strain BL21 using the pET expression system (Novagen, Madison, WI, USA) according to standard protocols. Protein purification was performed using precipitations and DEAE anion exchange chromatography. The purity of the protein samples was analyzed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970) and Coomassie Blue staining (Ausubel et al. 1989).

4.2 Site-directed mutagenesis and characterization of the salt bridge mutant proteins (III)

The mutations were introduced to the T. brucei brucei TIM cDNA using the QuikChange or ExSite site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. The following oligonucleotides containing the desired mutation were used in the polymerase chain reaction: 5’-AGCTGGGTGAGCAGCAAGATTGGA-3’ and 5’-AGCGATG- AGTGCGTGGGCTTCCTG-3’ for the R191A mutation, 5’-GCCCACGCAC- TCATCAGCAGCTGGGTGAGCAGC-3’ and 5’-GCTGCTCACCCAGCTGCTG- ATGAGTGCGTGGGC-3’ for the R191S mutation, 5’-GCCCACGCACTCAT- CAAGAGCTGGGTGAGCAGC-3’ and 5’-GCTGCTCACCCAGCTCTTGATG- AGTGCGTGGGC-3’ for the R191K mutation, 5’-CCAACAGCGAGCCGTCA- ACGGC-3’ and 5’-GCCGTTGACGGCTCGCTGTTGG-3’ for the D227A mutation, and 5’-CCAACAGCGAAACGTCAACGGC-3’ and 5’-GCCGT- 48

TGACGTTTCGCTGTTGG-3’ for the D227N mutation. Circular dichroism (CD) spectroscopy was used to study the thermal unfolding of the mutants R191A, R191K, R191S, D227A, and D227N. The melting curves were measured using a Jasco J-715 spectropolarimeter (Jasco, Tokyo, Japan) at a wavelength of 222 nm.

4.3 Crystallization (I-IV)

Prior to crystallization, the purified protein samples were concentrated to suitable concentrations, varying from 3 to 20 mg/ml, by centrifugal ultrafiltration in 10 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7.5, containing 25 mM NaCl, 1 mM dithiothreitol 1 mM ethylene diamine tetraacetic acid, and 1 mM NaN3. Initial crystallization conditions were screened using factorial screens (Zeelen et al. 1994) at room temperature (22°C) or 4°C. Crystals were grown in hanging drops containing equal amounts (1-5 µl) of protein and well solution. The optimization of the crystallization conditions was performed by altering the pH, protein and precipitant concentration, and different additives. Diffraction-quality crystals grew in times varying from a few days to several months. The ligands used were either added to the drop at concentrations of 5-10 mM beforehand or soaked into the crystal afterwards.

4.4 X-ray diffraction data collection and processing (I-IV)

The X-ray diffraction data were collected either on a MAR345 image plate detector (MarResearch) mounted on a Nonius FR591 rotating-anode X-ray generator at the Department of Biochemistry, University of Oulu (I, II), or the EMBL beamlines X11 (IV) and BW7B (IV) at DESY, Hamburg, or the beamline I711 at MAXLab, Lund, or a MARCCD detector (MarResearch) on the EMBL beamline X13 at DESY, Hamburg (III). The data were either collected at room temperature, in which case the crystal was mounted in a glass capillary, or at 100 K, in which case the crystal was soaked briefly in a solution containing mother liquor and cryoprotectant and flash-frozen in the cryostream. The atomic-resolution data (IV) were collected in three passes to get the maximum resolution at both the low and high resolution edges. All other data were collected in a single pass. All data were processed and scaled using Denzo and Scalepack (Otwinowski & Minor 1997). Further data reduction was done either with programs of the CCP4 program package (Collaborative Computational Project Number 4 1994) or with SHELX’97 (Sheldrick & Schneider 1997). All data processing statistics can be seen in the corresponding published articles. 49

4.5 Structure determination, refinement, and validation (I-IV)

4.5.1 Molecular replacement (I-IV)

All the structures were solved by the molecular replacement method (Rossman 1972, Rossman & Blow 1962) with the program AMoRe (Navaza 1994) using data from 8 to 4 Å resolution. Suitable search models either from the PDB or previously solved related structures from this study were used, omitting ligands, active site residues, and other possible sources of model bias.

4.5.2 Refinement of the medium-resolution structures (I-III)

The structures of the R191S mutant of T. brucei TIM at 1.65-Å resolution and E65Q mutant of L. mexicana TIM complexed with PGA and IPP at 2-Å resolution were refined against maximum likelihood targets using restrained isotropic refinement in Refmac or Refmac5 (Murshudov et al. 1997) from the CCP4 suite. For the 1.65-Å resolution structure of the R191S mutant, torsion, libration, and screw-rotation (TLS) parameterization (Schomaker & Trueblood 1968) was used as part of Refmac5 (Winn et al. 2001). Water molecules were built partly automatically with the program ARP/wARP (Perrakis et al. 1999) and partly manually. The program O (Jones et al.1991) was used for manual model building. Procheck (Laskowski et al. 1993) was used to validate the geometry of the final models. The statistics of the final models can be seen in the corresponding published articles. The structure factors and coordinates have been deposited at the PDB at the Research Collaboratory for Structural Bioinformatics with PDB entry codes 1QDS (the 2-Å resolution structure of the TIM-PGA complex), 1IF2 (the 2-Å resolution structure of the TIM-IPP complex), and 1KV5 (the 1.65-Å resolution structure of the T. brucei R191S mutant).

4.5.3 Refinement of the atomic-resolution structure (IV)

The refinement of the 0.83-Å resolution structure was carried out using SHELX’97 (Sheldrick & Schneider 1997) adding the high-resolution data gradually as the refinement proceeded (Table 5). As a starting model, a structure that had been refined against a 0.9-Å dataset was used; this structure was initially refined with Refmac and in the later stages with SHELX’97 to R and Rfree values of 12.05 and 13.85, respectively. In the beginning, the default geometric restraints and weighting scheme were used. Model-building was done with O (Jones et al. 1991). A test set of 2 % of all reflections was used to calculate Rfree. During the first 6 cycles, the model was refined isotropically. At this stage, water molecules were added to the structure, but other solvent molecules or 50

Table 5. Course of the refinement of the 0.83-Å resolution structure of the L. mexicana E65Q TIM complexed with PGA. ______Round R (%) Rfree (%) Number of Number of Comments parameters reflections ______0 30.13 27.44 1877 8153 Rigid body refinement (25-2.5 Å) 1 27.04 28.57 7475 39354 Isotropic B (25-1.5 Å) 2 20.68 20.71 8289 100611 PGA, 191 H2O (25-1.1 Å) 3 21.27 21.02 8265 184192 (25-0.9 Å) 4 20.02 20.26 9425 236819 482 H2O (25-0.83 Å) 5 20.03 20.29 9405 236819 477 H2O 6 16.80 17.54 9491 236819 1 acetate, 1 glycerol, 468 H2O 7 11.94 13.46 21532 236819 Anisotropic B, 492 H2O 8 11.83 13.60 21753 236819 13 alternative side chains 1 acetate, 2glycerols, 559 H2O 9 11.47 13.13 22458 236819 More rebuilding 10 11.54 13.01 22345 236819 33 alternative side chains 11 11.23 12.64 23113 236819 560 H2O 12 11.19 12.52 23152 236819 39 alternative side chains 590 H2O 13 11.14 12.51 23152 236819 More rebuilding 14 11.04 12.44 23680 236819 42 alternative side chains 561 H2O 15 10.96 12.56 23935 236819 Alternative water structure 2 acetates, 3 glycerols 16 9.83 11.28 23917 236819 Hydrogen atoms at riding positions 17 9.95 11.14 23885 236819 More rebuilding 18 9.64 10.91 23953 236819 More rebuilding 19 9.59 10.85 23954 236819 More alternative waters, loosening the restraints on PGA 20 9.50 10.88 23984 236819 Removing the restraints on PGA, loosening the restraints on protein atoms 21 9.49 10.85 29472 236819 Further improvement of the water structure 22 9.49 - 23983 241678 Including all reflections ______51 disordered residues were not modeled. The following cycles of refinement were performed anisotropically, and a detailed description of the model with disordered side chains and solvent was built. Most hydrogens were clearly visible in the Fo-Fc maps after moving to anisotropic refinement, and they were added to the model at the 16th cycle of refinement (Table 5). For the last cycles, the geometric restraints were released for the protein part and removed for PGA. The refinement was continued until the highest peaks in the Fo-Fc map were below 0.5 e-/Å3; the root mean square deviation of the final Fo-Fc map is 0.07 e- /Å3. At this stage, the highest peaks were due to disordered solvent or side- chain atoms not located close to the active site. The final crystallographic R and Rfree values were 0.0949 and 0.1085, respectively. The steps of the refinement are summarized in Table 6. As the very last step, all the restraints were removed, and a cycle of full matrix least squares refinement was carried out to calculate standard deviations for the atomic positions and bond lengths. The structure factors and coordinates have been deposited at the PDB at the Research Collaboratory for Structural Bioinformatics (PDB entry code 1N55).

4.6 Structural comparisons (I-IV)

To identify highly conserved residues in TIM, an amino acid sequence alignment of all 60 TIM sequences available was performed with ClustalX (Thompson et al. 1994, Jeanmougin et al. 1998). For structural comparisons, the threedimensional structures were superimposed within O (Jones et al. 1991), using the Ca atoms of each of the b strands and a helices of the TIM barrel. 5 Results

5.1 Structure of the Leishmania mexicana triosephosphate isomerase superstable E65Q mutant (I)

The 2-Å resolution crystal structure of the E65Q mutant at room temperature shows that, as in T. brucei TIM, the Gln65 side chain is completely buried at the dimer interface. There are essentially no structural changes between the wild type L. mexicana enzyme and the E65Q mutant, including the water structure near the Gln65 side chain. The Gln65 side chain is in an optimal hydrogen- bonding scheme. The Ne2 protons are engaged in hydrogen bonds to the main chain oxygen atoms of Lys13 in loop 1 and Ala42 in loop 2 of the same subunit. These hydrogen bonds are present in all other TIM structures, except for the wild type L. mexicana TIM which has the glutamate instead of the glutamine at this position (Williams et al. 1999). This hydrogen-bonding network explains the increased stability of the E65Q variant. The E65Q crystals grow very large (longest dimension up to 1 mm). The crystals are very solid and are not very radiation sensitive. Possibly, the superstability of the E65Q mutant favors the growth of such large and solid crystals. When frozen in the presence of 20-25 % glycerol, they diffract X-rays to approximately 0.8-Å resolution. Furthermore, the quality of these crystals can be improved by repeatedly unfreezing and refreezing them. After such handling, decreased mosaicity and increased diffraction can be observed. One more advantage of this crystal form is that the flexible loop 6 is not involved in crystal contacts, and thus, the ligand can be exchanged in the crystalline state.

5.2 Comparison of the binding mode of 2-(N-formyl-N-hydroxy)- aminoethyl phosphonate and phosphoglycolohydroxamate (II)

The structure of the TIM-IPP complex, where IPP was soaked into the crystal to replace PGA, was refined at 2-Å resolution. According to the electron density, 53 the mode of binding of IPP is well defined, and at this resolution, no residual electron density for PGA can be seen. The hydroxamate moiety is bound in a planar conformation. Two interesting differences can be pointed out when comparing the mode of binding of IPP to L. mexicana TIM and the binding of PGH to chicken TIM. Firstly, the hydroxamate planes do not superimpose. In the IPP structure, the proximal part, atoms C1 and O1 are pointing further down, towards the Asn11 side chain. The largest displacement is seen for the O1 atom which has moved by approximately 1 Å. Secondly, the side chain of the catalytic glutamate has adopted a different orientation in these structures. The equivalent carboxylate oxygen atoms have also moved by approximately 1 Å. The O1 atom of IPP is at a hydrogen-bonding distance to Nd2 of Asn11 and to Ne2 of His95. The carboxylate oxygens of Glu167 are near the hydroxamate plane. Oe2 of Glu167 is near N2, and Oe1 of Glu167 is near O2. The O2 atom of IPP is also hydrogen bonded to Nz of Lys13 and Ne2 of His95. In the TIM-PGH complex, the equivalent Glu167 carboxylate atoms are near N1 and O1. In both complexes, there are hydrogen bonds from Ne2 of His95 to O1 and O2 of the ligand. As in the IPP complex, O2 of PGH is also hydrogen bonded to Nz of Lys13. The hydrogen-bonding scheme of the ligands with the catalytic residues Glu167, His95, Lys13, and Asn11 in the TIM-IPP and TIM-PGH complexes is depicted in Fig. 5 of the published article II. In both the TIM-IPP and the TIM-PGH structures, the His95 side chain has no hydrogen bonding interactions with water. Nor do the hydroxamate atoms of IPP and PGH contact any water molecules. Nz of Lys13 is hydrogen bonded to one water molecule. Through this interaction, a water-mediated hydrogen bond with the phosphate moiety of the ligand is formed. In both structures, Nz of Lys13 contacts the bridging atom {C4(IPP)-Nz(Lys13) = 3.2 Å and O4(PGH)-Nz(Lys13) = 3.4 Å}. Furthermore, in both complexes, there is a cluster of three water molecules near the Glu167 side chain. From the superposition of the available TIM-ligand complex structures, it can be seen that there are two preserved anchoring interactions: (a) the phosphate moiety and (b) atom O2 of the ligand. In all liganded TIM structures, the phosphate moiety is bound in the same orientation; it is always hydrogen bonded to seven peptide NH-groups, distributed over loops 6, 7, and 8. The O2 atom is hydrogen bonded to Nz of Lys13 and Ne2 of His95. In this mode of binding, the O2 atom of the substrate is fixed with respect to the bridging oxygen atom, O4. The near cis arrangement for the c3 torsion angle of the bridging oxygen atom and the O2 atom of the substrate is an essential feature of the TIM active site geometry, as it is expected to prevent the undesirable phosphate elimination side reaction. The Glu167 side-chain displays large variation in its position in different structures. In contrast, the other catalytic residues, in particular Lys13 and His95, always adopt the same conformation. The Glu167 side chain occurs either in a swung-out position (in the unliganded structure) or in a swung-in position (in the liganded structure) (Lolis & Petsko 1990, Noble et al. 1993). In the swung-out conformation, Glu167 is hydrogen bonded to the peptide N and Og of Ser96. In addition, there is a hydrogen bond between Oe2 of Glu167 and a water molecule. In this 54 conformation, the Glu167 side chain Oe2 atom also contacts Sg of Cys126. The Sg atom of Cys126 is also close to the main chain O atoms of Ile124 (at 4.3 Å distance) and Leu93 (at 3.8 Å distance), suggesting that the Sg proton of Cys126 is hydrogen bonded to these two peptide oxygens. Therefore, the Oe2(Glu167)- Sg(Cys126) contact is a van der Waals contact. Interestingly, in the swung-in conformations, the van der Waals contact with the Sg of Cys126 is maintained. However, the two hydrogen bonds to Ser96 are lost. They are not replaced by hydrogen bonds to any other protein atoms. In the conformational switch from the swung-out to the swung-in conformation, the Glu167 side chain rotates towards the ligand and towards Leu232. For example, in the TIM-IPP and the TIM-PGH complexes, there is a contact between O of Leu232 and the Glu167 side chain. From the geometry of the active site, this contact seems to be a van der Waals contact. The movement of the Glu167 side chain is only possible in the closed conformation of loop 7, since in the open conformation, it would bump into the main chain oxygen of Gly211. There are two structured waters in the region vacated by the Glu167 side chain as it moves towards its new position. Together with the water molecule present in the unliganded structure near Oe2 of Glu167, these two water molecules form the cluster of three water molecules described previously (Zhang et al. 1999). Therefore, in the IPP-swung-in, as well as in the PGH-swung- in conformation, the Glu167 side chain makes no hydrogen bonds with the protein, one hydrogen bond to a water, and one hydrogen bond to the ligand N- hydroxyl group. In the TIM-IPP structure, the Oe1(Glu167)-O2 distance is 3.3 Å and in the TIM-PGH structure, the Oe1(Glu167)-O1 distance is 2.6 Å. The observed conformational differences of the Glu167 side chain in the two swung-in conformations of the TIM-IPP and TIM-PGH complexes correlate with the relatively high B-factors of this side chain in the liganded structures; it is also seen that the average B-factors of the Glu167 side chain and the interacting ligand are similar. In the unliganded structure, the Glu167 side-chain B-factors are in the same range as the B-factors of Lys13 and His95. These side chains are well hydrogen bonded to the rest of the protein. In all liganded and unliganded structures, the Lys13 side chain has low B-factors and forms a salt bridge to the Glu97 side chain. For the His95 side chain, a strong hydrogen bond is observed from Nd1 to N of Glu97 in all structures, suggesting that this histidine is neutral: unprotonated at Nd1 and protonated at Ne2.

5.3 Mutagenesis and structural studies on the role of the conserved salt bridge between Arg191 and Asp227 (III)

Four salt bridge mutants that partially or completely abolish the interaction between the side chains of Arg191 and Asp227 in T. brucei brucei TIM were constructed and purified. These salt-bridge forming residues reside at the backside of the molecule, connecting the C-terminus of helix 6 to the C-terminus of helix 7. All the mutants appeared to be less soluble than the wild-type enzyme 55 during purification. Furthermore, the R191A variant is mainly found in inclusion bodies, indicating that its folding properties have been severely affected by the mutation. CD-spectra and thermal denaturation curves were measured for all of the mutants. In addition, the crystal structure of the R191S variant complexed with PGA was determined at 1.65-Å resolution. According to the Tm values, the overall stability of the mutants has somewhat decreased compared to the wild- type enzyme. The largest difference is seen for the R191A mutant, for which the Tm value has decreased by 5°C. In the fairly high-resolution crystal structure of the R191S mutant, the side chain of serine 191 is well defined. Hydrogen bonds of the Og atom of the serine side chain to water molecules have partly compensated for the loss of the salt bridge. Other hydrogen bonding contacts in the area have also been preserved by this water-mediated network. Apart from the mutation and the new water molecules, there are no structural changes in the area. In the R191A variant, the hydrogen bonding network has to deviate more from the wild-type enzyme, due to the lack of a suitable hydrogen bond donor or acceptor in the side chain at position 191. The results of this study indicate that the conserved salt bridge and the hydrogen-bonding interactions within are a requirement for efficient folding of TIM

5.4 The atomic-resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2- phosphoglycolate (IV)

5.4.1 Quality of the model

The model of the TIM-PGA complex was refined to 0.83-Å resolution. Default geometric restraints were applied in the beginning of the refinement, and they were released for the protein part and removed for the ligand towards the end. The very last cycle that was used for calculating the estimated standard deviations of the model was done unrestrained. 94.1 % of the residues are in the most favourable region and 5.9 % in the allowed region in the Ramachandran plot (Ramachandran et al. 1963), including the catalytic Lys13. Standard deviations of bond lengths and angles, for instance, are larger than normally in average resolution structures, in which tight geometric restraints are used, but well in agreement with other structures at atomic resolution. Features, such as triple-conformations of side chains, alternative hydrogen-bonding networks involving protein and solvent atoms, and hydrogens, can be seen in the electron density maps. Based on the last full-matrix least squares refinement cycle, the estimated standard deviations of bond lengths, for instance, range between 0.02 and 0.03 Å, which allows for very high precision in determining, for example, if a certain bond has more single or double bond character. 56

5.4.2 Structural heterogeneity

Due to the high resolution of the diffraction data, many of the disordered residues could be modeled with confidence. Altogether, 42 of the 250 residues have been built in more than one conformation. Nevertheless, some of the solvent-exposed side chains had to be considered as highly disordered with no preferred conformations. Those side chains have been included in the model in one or two conformations, depending on the electron density, but sometimes there was residual electron density in the surroundings that could not be explained by the model. Mostly, the alternative conformations occur in the side chains, but in a few cases, also the main chain is disordered. For example, the tip of the flexible loop 6 (residue Gly175) has two distinct conformations. In the major conformation (60 % occupancy), the peptide proton of N(Gly175) points to Og of Ser213, providing a reasonable hydrogen-bonding interaction (Og(Ser213)-N(Gly175) distance is 3.29 Å). In the minor conformation, this interaction is weakened and Gly175 is unfavorably close to Og of Ser213; for instance, the distance Ca (Gly175)- Og(Ser213) is 3.21 Å. This disorder seems to reflect the intrinsic loop-opening properties of loop 6, which is also visualized by the anisotropic B-factors of residues adjacent to Gly175 (Thr174 and Lys176). In the active site, PGA is bound in two conformations. In both of them, the O11 atom of PGA is hydrogen bonded to Oe2 of Glu167. In the predominant conformation (70% occupancy), O12 of PGA is at a hydrogen-bonding distance to Nz of Lys13 and Ne2 of His95, while in the minor conformation (30% occupancy), it points further down and is weakly hydrogen bonded to Ne2 of Asn11. In the predominant conformation, the Ne2(Asn11)-O12(PGA) distance is 4.17 Å, while in the minor conformation this distance is 3.22 Å. The difference between the two conformations concerns a rotation about the C1-C2 bond of PGA. The torsion angle O1P-C2-C1-O12 is -35° for the major conformation and 0° for the minor conformation. The Glu167 side chain is also twofold disordered. The disorder concerns the position of Oe1, whereas Oe2 adopts always the same position, being hydrogen bonded to O11 of PGA. The structural disorder of the carboxylate moieties of PGA and the Glu167 side chain appears not to be a concerted motion - instead, the active-site geometry suggests that these two groups move independently of each other, while keeping the O11(PGA)- Oe2(Glu167) hydrogen bond intact. In the major conformation of Glu167 (80 % occupancy, c1=-52, c2=-169, c3=- 66), the Oe1 atom points to Sg of Cys126 at a distance of 4.12 Å, while in the minor conformation (c1=-50, c2=-161, c3=-53), Oe1 is rotated away from Sg of Cys126 at a distance of 4.69 Å. This structural disorder appears to be correlated with heterogeneity of Sg of Cys126, as there exists a minor conformation in which this atom is rotated into the space created by the rotation of Oe1 of Glu167. In both Glu167 side-chain conformations, the Oe1 atom is within a hydrogen-bonding distance from O11 of PGA as well as Wat28. This water is the first water of a network between Glu167 and bulk solvent. It consists of waters 28, 114, 38A, and 58. Wat58 is also hydrogen bonded to Og of Ser96. There also 57 exists an alternative arrangement for this network, consisting of waters 28, 114, 38B, 107, and 58.

5.4.3 Anisotropy

The large amount of data at this high resolution allowed for expanding the description of the model to anisotropic displacement parameters. The anisotropic description was applied to all atoms, including solvent molecules, which caused a significant drop in both R and Rfree (Table 5). As seen in other structures at atomic-resolution, none of the atoms agree with the traditional isotropic description. As examined by PARVATI (Merritt 1999) and Rastep (Merritt & Bacon 1997), the mean anisotropy of all protein atoms in the structure is 0.53 (with a s of 0.1) deviating significantly from the value 1, which means perfect isotropy. The anisotropy value refers to the ratio of the shortest and longest principal axis of the thermal ellipsoid describing the anisotropic scattering properties of an atom (Merritt 1999). The mean isotropic B of the protein atoms is 11.8 Å2. In this crystal form, the dimer twofold axis coincides with a crystallographic axis and consequently, the molecular center is on this twofold axis. The analysis of the mean anisotropy of the protein atoms of the dimer as a function of the distance from the dimer center shows the least anisotropy at the center (with a mean anisotropy of 0.9), which increases smoothly to a mean anisotropy of 0.3 at the extremeties (25 Å away from the center). Consistently, very low anisotropy is seen at the C-termini of the b strands. This correlates with the low isotropic B- factors of these regions. The regions with the highest anisotropy are in the TIM- barrel framework a helices, in particular at their C-termini. In the active site, the side chain of His95 shows significant anisotropic behavior with an anisotropy value of 0.47 for Ne2. This anisotropic movement occurs in the plane of the imidazole ring. The side chains of Lys13 and Asn11 are less anisotropic with anisotropy values of 0.63 for Nz of Lys13 and 0.62 for Nd2 of Asn11. Glu167, which is present in two conformations, displays significant anisotropy in the minor conformation with anisotropy values of 0.59, 0.41, 0.37, and 0.54 for Cg, Cd, Oe1, and Oe2, respectively. For the major conformation, the corresponding anisotropy values are 0.66 (Cg), 0.87 (Cd), 0.50 (Oe1), and 0.43 (Oe2). The mean anisotropy of PGA is 0.47 and 0.43 for the major and minor conformation, respectively. It is interesting to note the relatively high anisotropy of PGA and the side chain of the catalytic glutamate, despite the fact that the main chain regions defining the active site have relatively low anisotropy as well as low isotropic B-factors. For example, the anisotropy of the Ca atoms of Lys13, His95, Asn11, and Glu167 are 0.86, 0.77, 0.84, and 0.69, respectively. The anisotropy of the flexible loop 6 has a direction parallel to the loop opening/closure event. The anisotropy is more pronounced at the C-terminal end of the loop, concerning residues Gly173-Ala178. The most anisotropic part concerns residues Thr174 and Gly175 when only the main chain atoms are 58 considered, whereas the side chain of Lys176 has one of the highest anisotropy values in the whole structure (0.10 for the Cb atom).

5.4.4 Active-site geometry

The phosphate moiety of PGA is hydrogen bonded to main chain N atoms from loops 6, 7, and 8, while the triose moiety forms hydrogen bonds to side chain atoms, in particular Lys13, His95, and Glu167. The phosphate moiety is also hydrogen bonded to the protein via four water molecules: via Wat44 to Lys13 and via Wat186, Wat8, and Wat1 to loops 7 and 8. Wat1 is buried between the ligand and protein and is not part of the network of water molecules that extends to the bulk solvent. Wat1 is hydrogen bonded to N of Gly212, O2P of PGA, and O of Leu232, and it is at 3.69 Å distance from C2 of PGA. The side chains of Lys13 and His95 form strong hydrogen bonds to the bulk of the protein. Nz of Lys13 is tightly fixed to its position by three hydrogen bonds to Oe1 of Glu97, O2 of PGA, and Wat45. Ne2 of His95 is hydrogen bonded to O12 of PGA (2.74 Å), whereas the side chain position of His95 is stabilized by a hydrogen bond between Nd1 and N of Glu97 (2.96 Å). The Glu167 side chain is only weakly interacting with the rest of the protein. The Glu167 side chain carboxylate oxygen atoms span the region between Ca of Gly211 (at 3.39 Å from Oe2), O of Leu232 (at 3.60 Å from Oe2), and Sg of Cys126 (at 4.12 Å from Oe1). The hydrogen atom on Sg of Cys126 points towards the main-chain oxygens of 124 and 93, as is also confirmed by the residual positive density in the Fo-Fc map; therefore, the Oe2(Glu167)-Sg(Cys126) is a van der Waals contact. Oe1 is at a distance of 3.29 Å from Ne2 of His95, but the geometry does not allow for a good hydrogen bond. Two other fully conserved side chains (Ile172 in loop 6 and Leu232 at the beginning of loop 8) also contact the Glu167 carboxylate group. None of these contacting protein atoms allow for good hydrogen bonding. Except for the ligand, the only observed hydrogen-bonding partner of the Glu167 side chain is Wat28. This hydrogen bond exists in both the major and the minor conformation of the Glu167 side chain with distances of 2.71 and 2.80 Å. In both the major and the minor conformation, there exists a short (2.61/2.55 Å) hydrogen bond between Oe2 of Glu167 and O11 of PGA. The Cd-Oe2 and Cd- Oe1 bond distances in Glu167 are 1.26 and 1.19 Å, respectively, whereas the C1- O11 and C1-O12 bond distances are 1.22 and 1.19 Å, respectively. These distances are in the range of the C-O bond distances of the carboxyl moiety of the fully protonated crystal structure of PGA (Lis 1993) which are 1.23 Å for the C=O double bond and 1.31 Å for the C-OH single bond. In the trianionic species of PGA, the C-O distances of the carboxylate group are 1.24 and 1.26 Å (Lis 1993). The active site is at the dimer interface, and the well-defined conformations of Asn11 and Lys13 are stabilized due to the tight interactions with loop 3 of the second subunit; for example, Thr75 at the tip of this loop is hydrogen bonded to Nd2 of Asn11 and Oe2 of Glu97, which forms a salt bridge to Nz of Lys13. 59

Analysis of the B-factors shows that the tip of this loop is very rigid (BCa Gly76=5.4 Å2) and the anisotropy is very low (ACa Gly76=0.82). 6 Discussion

6.1 The role of the conserved salt bridge Arg191-Asp227 in folding and stability (III)

The removal of the salt bridge between Arg191 and Asp227 causes subtle changes in the stability and catalytic properties of TIM, especially in the case of the most disruptive R191A mutant. In particular, though, the (re)folding kinetics of the mutants are deteriorated. Assuming that the structural differences are minimal, as can be seen for the R191S mutant, the effect on catalysis is intriguing as the hydrogen bonding network is 20 Å away from the active site. Its importance for catalysis would be consistent with the full evolutionary conservation of the salt bridge, suggesting that some long distance effects, such as electrostatics (Fehrst 1999) or dynamics (Wand 2001) are important for obtaining optimal active site properties. The salt bridge and the related hydrogen bonding network anchor the C-terminal ends of helices 6 and 7 to each other. However, from the present data, it is not clear whether this could influence the dynamical properties of the flexible loops 6 and 7 involved in substrate binding. Although the R191A mutant has reduced catalytic activity compared to the wild-type T. brucei TIM, it has to be stated that the kinetic constants fall within the range reported for several TIMs from different organisms. It therefore seems unlikely that maintaining the activity is the sole evolutionary pressure for the conservation of this specific interaction. The salt bridge is close to the surface of the protein, but it is, nevertheless, almost completely buried. The amount of charge at this region varies from organism to organism. On one side of the salt bridge, there is always one positively charged group - arginine, or in some cases lysine. On the other end, there can be either one or two negatively charged groups - the DX(D/N)G motif. Thus, it does not seem likely that changing the charge in this area would be the reason for the loss of enzyme activity. Indeed, mutating both partners of the salt bridge also results in a non-functional enzyme (Silverman et al. 2001). An increasing number of experimental studies (Morgan et al. 2000, Benítez- Cardoza et al. 2001) and theoretical analyses (Mirny & Shakhnovich 1991, 61

Kannan et al. 2001) address the folding properties of TIM and other TIM barrel enzymes. Computational approaches have pointed out the importance of folding initiation sites (Mirny & Shakhnovich 1991), and Gly230 in the DX(D/N)G motif has been postulated to be such a nucleation site (Kannan et al. 2001). This motif is located at the extremity of the dimer, and it tethers the C-terminal a 7b7a 8b8 unit to the rest of the protein via an extensive hydrogen bonding network. Also the N-terminus participates in this hydrogen bonding scheme. Apparently, the formation of the Arg191-Asp227 salt bridge in this network is important for efficient folding. This would also explain the low expression yields and solubility problems of the mutants in this study, as well as the loss of functional enzyme in an earlier mutagenesis study (Silverman et al. 2001). Furthermore, the recovery of activity after refolding is much less effective for the mutants than for the wild-type enzyme. It is interesting to point out that refolding experiments with two monomeric TIM-barrel proteins, TrpF (Eder & Kirscher 1992) and TrpA (Zitzewitz & Matthews 1991), have shown that, in these proteins, two folding units exist; the ab units 1-6 fold first and subsequently assemble with the ab units 7 and 8 at the end of the folding pathway. In a recent computational study on the van der Waals contacts in different protein folds, tight van der Waals clusters were identified, and their relevance for folding was discussed (Berezovsky & Trifonov 2001). In TIM, there exists a large ”van der Waals lock” that is composed of nine discontinuous sites along the whole protein. One of the regions involved is situated close to the salt-bridge region, where a hydrophobic cluster was identified in this study, as well. This hydrophobic core could be one of the major factors that stabilize the fold. The results obtained from the present study on the TIM salt bridge variants indicate that the evolutionary pressure for the conservation of the salt bridge is due to the requirement for efficient folding of TIM.

6.2 Superstability of the E65Q mutant (I)

Often highly stable enzymes, for example from thermophilic species, have reduced catalytic activity at normal temperatures, because the stabilizing features introduce extra rigidity to the structure (for review, see Jaenicke 2000). In the context of engineering for instance industrially important enzymes, it would be of great interest to develop highly stable enzymes that would still have high catalytic power. L. mexicana TIM has a Glu at a position at the dimer interface, where all other TIMs have a Gln (Kohl et al. 1994). The amide protons of the Gln are hydrogen bonded to the main chain oxygens of Lys13 and Ala42. Burying the ionizable Glu side chain in an uncharged environment is expected to have a destabilizing effect. Yet, L. mexicana TIM is as stable as, for instance, T. brucei TIM, which shares 68 % sequence identity with the L. mexicana enzyme (Kohl et al. 1994). Thus, there must exist other interactions that compensate for the loss of stability at this position. Mutating this Glu to Gln results in a superstable enzyme, with 62

Tm of 83°C at neutral pH, compared to the 56°C of the wild type enzyme. Despite this enormous gain in stability, the mutant enzyme has similar catalytic properties as the wild-type enzyme (Williams et al. 1999). The crystal structure of the wild type L. mexicana TIM shows that, like the Gln in all other structures, the Glu side chain is buried at the dimer interface, pointing towards the active site of the same subunit (Williams et al. 1999). Also the water structure at this region is identical to other structures. The carboxylate group is expected to be protonated and seems to be optimally hydrogen bonded to the main chain O of Lys13. The stability of the L. mexicana TIM is dependent on pH, because of the ionization of the carboxylate group, which has a pKa of about 7 in this buried environment. From the structure, it is not totally clear what are the additional stabilizing features in the L. mexicana TIM. Compared to T. brucei TIM, there are somewhat fewer glycines and more prolines. In the vicinity of the Q65E substitution, there is also a substitution of a Ser to Pro, which is expected to contribute to the stability. The mutation E65Q, besides giving a highly thermostable enzyme, abolishes the pH-dependency of the thermal stability of the enzyme. From the structure of the E65Q mutant (I), it could be seen that no structural rearrangements have taken place due to the mutation. It is intriguing that a point mutation can cause such a dramatic improvement in the stability of an enzyme - especially since it concerns only a substitution of one oxygen atom with a nitrogen. The increased stability of this mutant is due to an extra hydrogen bond formed by the Gln side chain in each subunit as well as the removal of an ionizable residue from the uncharged dimer interface. It is remarkable, that this stabilization affects only the dimer and not the monomer, even though the additional hydrogen bond is formed within one subunit. The explanation for this could be that the hydrogen bond between Ne2 of Gln65 and the main chain O of Ala42 is only formed as the subunits associate to form the dimer. Indeed, this region and the hydrogen-bonding contacts of Gln65 are seen to be different in the monomeric TIM variants, where Gln65 is only hydrogen bonded to the main chain O of Trp12 and Od1 of Asn11 via Ne2. The E65Q mutant crystals diffract X-rays to exceptionally high resolution for protein crystals - up to 0.8 Å. This is largely due to the large size of these crystals, they reach the volume of approximately 0.15 mm3 in a few days. The solvent content is average, approximately 50 %. It is likely that the superstability of these crystals also affects the crystallization properties. Furthermore, these crystals are unusually insensitive for all kinds of handling and radiation. On a rotating anode X-ray source, data from unfrozen crystals can be collected for at least three days without any visible effect on the diffraction quality. The crystal contacts are such that the flexible loop 6 is not tied to its position, which enables changing the ligand bound in the crystalline state. This feature was succesfully utilized in solving the structure of the TIM-IPP complex. Despite the increase in diffraction when frozen, the frozen crystals display also rather high mosaicity (0.5-0.8°). Often, this can be be improved by repeatedly unfreezing and refreezing the crystal in the cryostream, as has also been noted for other protein crystals (Yeh & Hol 1998). 63

6.3 The active site (II, IV)

6.3.1 The catalytic role of Asn11

Theoretical calculations have pointed towards a possible role for Asn11 in the reaction mechanism of TIM (Alagona et al. 1995, Peräkylä & Pakkanen 1996), in providing a positive electrostatic potential and stabilizing a reaction intermediate or transition state. In the known X-ray structures of TIM-ligand complexes, however, only weak hydrogen bonding between Asn11 and the ligands is seen. In the structure of the TIM-IPP complex from this study, there is a strong hydrogen bond between O1 of IPP and Nd2 of Asn11. IPP is an analogue of the substrate DGAP, and the O1 atom is equivalent to the aldehyde oxygen atom. The TIM-IPP structure therefore suggests that in the catalytic cycle, a small but significant rearrangement of the substrate also occurs. Upon binding of DHAP, an oxyanion hole formed by Lys13 and His95 facilitates the abstraction of the C1 proton of DHAP. Subsequently, the intermediate rearranges in the active site, such that O1 now fits well into a second oxyanion hole formed by Nd2 of Asn11 and Ne2 of His95. In this binding mode, the development of a negative charge on the atom O1 is stabilized, which facilitates the proton exchange at atom C2 and the formation of the product, DGAP. In the unliganded structure, a conserved well-defined water molecule is bound in this oxyanion hole 2, hydrogen bonded to Nd2 of Asn11 and Ne2 of His95. This water is displaced upon ligand binding. In the atomic-resolution structure of the TIM-PGA complex, PGA is seen to adopt two distinct conformations in the active site. The minor conformation has a very unfavourable c3 (O1P-C2-C1-O12) torsion angle of -2°. In this conformation, the O12 atom approaches the oxyanion hole 2, being at least weakly hydrogen bonded to Ne2 of Asn11. As it seems that this interaction is able to force the ligand to such an energetically costly conformation, the stabilizing power of this oxyanion hole during the catalytic cycle is likely to be significant.

6.3.2 Adaptability of the active site and the possible role of low- barrier hydrogen bonds in the catalytic mechanism

The reaction catalyzed by TIM is conceptually rather simple, as it only concerns shuttling of protons. The active site can be seen to consist of two parts: a binding pocket for the phosphate moiety and a catalytic site for the binding of the triose moiety. It is thought that during the catalytic cycle, the phosphate group of the substrate has to be held in plane with respect to the rest of the molecule in order to avoid phosphate elimination (Lolis & Petsko 1990). Therefore, mechanistically, the reaction becomes complicated. An additional difficulty that has to be overcome by the enzyme is that C-H bonds are very stable. For 64 example, the pKa of methane is estimated to be 40 (Cleland 1998). Consequently, under physiological conditions, a deprotonated carbon atom is very unstable. If the resulting carbanion is stabilized, for example in the presence of an adjacent carbonyl function, then the pKa is much lower, near 20 (Zhang et al. 1999). This is still considerably different from the pKa of a glutamate side chain, which is known to extract the proton from DHAP in the first step of the TIM reaction (Knowles 1991, Knowles & Albery 1977). Nevertheless, TIM is an extremely efficient catalyst. It is an intriguing question, what the properties are that enable the enzyme to do this difficult task in such a superior way. The active site is located at the C-termini of the TIM barrel b strands and in fact, residues from each of the strands (or subsequent loops) contribute to the active site. In addition, Thr75 from loop 3 of the opposing monomer is hydrogen bonded to the side chains of Asn11 and Glu97, thus providing essential stabilizing interactions. PGH and IPP are transition-state analogues mimicking structural features of the ketone (DHAP) and aldehyde (DGAP) substrate, respectively. The comparison of the mode of binding of these two compounds suggests that the precise details of the active site geometry concerning substrate binding change as the reaction proceeds from substrate to product. This is correlated with the structural differences between an a -hydroxyketone and an a -hydroxyaldehyde, as the planar carbonyl function is transferred from the C2 position to the C1 position. In particular, it has been shown that the side chains of Lys13 and His95 create a stabilizing oxyanion hole for the ketone carbonyl group, whereas Asn11 and His95 form such an oxyanion hole for the carbonyl group of the aldehyde substrate. In the apo structure, a water molecule is bound in the latter pocket. In the atomic-resolution structure, the active site of TIM is complexed with a different transition-state analogue, PGA. It is observed that the triose moiety of PGA occurs in two conformations, while the phosphate moiety is bound in a well-defined single conformation. The P-O1P (O1P is bonded to C2) distance is 1.62 Å, whereas the other P-O distances are 1.49 Å, 1.50 Å, and 1.55 Å. The corresponding distances in the crystallized trianionic species of PGA are 1.64 Å, 1.51 Å, 1.50 Å, and 1.53 Å, respectively (Lis 1993). The phosphate is hydrogen bonded to main-chain nitrogen atoms from loops 6, 7, and 8. The triose moiety is hydrogen bonded to side chains only (Asn11, Lys13, His95, and Glu167). In addition, it is in van der Waals contact with the highly conserved side chains of Ile172 in loop 6 and Leu232 in loop 8. PGA is a transition-state analogue in which the methanol group of the ketone substrate (DHAP) is replaced by a hydroxyl group. The O12 atom mimics the ketone moiety, whereas the O11 atom mimics the C1 atom of DHAP from which Glu167 abstracts a proton. The O11 atom of PGA points to both the Oe1 and Oe2 of Glu167, implying that it interacts with the syn orbitals of this carboxylate moiety, which is the optimal geometry for proton abstraction (Lolis & Petsko 1990), as the syn orbitals are known to be more basic than the outward-pointing anti orbitals (Gandour 1981). In the major conformation, O12 of PGA binds to Nz of Lys13 and Ne2 of His95, whereas in the minor conformation, the O12 of PGA is bound at the expense of induced strain in the molecule (the C1-C2 dihedral is 65

0°), with low occupancy in the second oxyanion hole. The disorder of PGA in this structure correlates with the differences in the mode of binding of PGH and IPP, resembling the situation as the reaction proceeds from DHAP to DGAP. A key feature of the active site is a very short hydrogen bond between the carboxylate groups of PGA and Glu167. The O-O distance for the usual hydrogen bond between two oxygen atoms, with the hydrogen being firmly attached to one of them, is around 2.8 Å (Cleland et al. 1998), as can be seen also for most O-O hydrogen bonds in the atomic-resolution structure from this study. However, the distance between O11 of PGA and Oe2 of Glu167 is 2.61 and 2.55 Å, when considering the major and minor conformations, respectively. This is significantly shorter than a normal hydrogen bond and similar to the O-O distance expected for a LBHB (Cleland et al. 1998). The proton on Oe2 is not visible in the electron density maps. This may imply that the proton is indeed shared with the carboxylate groups of PGA and the glutamate, and is able to freely diffuse between these two atoms. NMR studies have shown that in the complex of TIM with PGH, there exists an LBHB between the NOH hydrogen and the catalytic glutamate (Harris et al. 1997). The formation of this LBHB requires somewhat different conformations of the Glu167 side chain and the ligand, as compared to the TIM-PGA complex. Clearly, the glutamate side chain and ligand geometry can flip between different conformations allowing for strong interactions between the glutamate and different atoms of the substrate. This apparently allows the side chain to shuttle protons directly between the two carbons of the substrate (as in the classical mechanism) as well as via the oxygen atoms (as in the criss-cross mechanism). The former, which explains the tritium incorporation from DHAP to DGAP (Knowles & Albery 1977), requires that the proton transfer between the oxygens is mediated by the His95 side chain (Lodi & Knowles 1991, Bash et al. 1991). In the atomic-resolution TIM-PGA complex, the Ne2 of His95 is hydrogen bonded to O12 of PGA (2.74 Å) and interacting also with O11 at a distance of 3.21 Å. The electron density suggests that Ne2 is protonated and Nd1 unprotonated. The anisotropic B-factors of His95 indicate that the histidine side chain moves in a concerted motion in the plane of the imidazole ring. When examining the mode of binding of IPP and PGH, it can be seen that such dynamical properties would facilitate proton transfer between the two substrate oxygen atoms via Ne2 of His95, as has been observed to occur (Lodi & Knowles 1991). In addition to the ligand, the Glu167 side chain is hydrogen bonded to a water molecule, which is part of a network that extends from the active site to the bulk solvent. In the atomic-resolution structure, this network is seen to occur in two alternative arrangements. The importance of these waters for catalysis has been discussed (Zhang et al. 1999). Proton exchange between the catalytic glutamate and bulk solvent via this network might also explain the low yield of tritium transfer from the labeled C1 of DHAP to C2 of DGAP during catalytic turnover (Rieder & Rose 1956, 1959, Bloom & Topper 1956, 1958, Knowles et al. 1972, Knowles & Albery 1977). The observed dynamical properties of the glutamate side chain and the water molecules could reflect active proton exchange during catalysis. 66

At about the same time with the atomic-resolution structure in this study, a fairly high-resolution (1.2 Å) structure of TIM complexed with the substrate DHAP was published (Jogl et al. 2003). In the active site, only DHAP in a single conformation is seen. Surprisingly, the substrate is not bound in the ”in-plane” conformation, which is expected to prevent the harmful phosphate elimination side reaction, but is seen to be in a relaxed conformation with the phosphate out of the plane of the C2 carbonyl group. On the other hand, recently, the in-plane conformation of transition-state analogues has been seen to occur also in the active site of methylglyoxal synthase (Saadat & Harrison 2000, Marks et al. 2001). This raises the question whether the planarity of the substrate has anything to do with preventing methylglyoxal formation. It must be noted that enzyme active sites are built such that they bind the transition states or intermediates tighter than the substrate or the product. Thus, it may well be that the conformation of the bound substrate (or product) does not have such a mechanistic relevance as the transition state or intermediate. On the other hand, the transition-state- resembling compounds are of course rather hypothetical, and it cannot be taken for granted that they represent the true reaction intermediates or transition states. It seems surprising that soaking the crystals on DHAP results in a structure with only DHAP bound - in a single, well-defined conformation. It is true that in solution, the equilibrium is strongly on the DHAP side. On the enzyme surface, the situation is different, but still it is expected that approximately 70 % of the species bound would be DHAP and 30 % DGAP (Belasco & Knowles 1980). It may be that as the resolution is only 1.2 Å, a minor species could have been invisible or uninterpretable. For sure, the results by Jogl et al. (2003) confirm that the equilibrium indeed is shifted to the DHAP side also in the active site of TIM. The active site of TIM is remarkably similar to methylglyoxal synthase (Cooper 1984), and both of these enzymes bind DHAP, even though they are not structurally or amino-acid sequencewise related (Davenport et al. 1991, Saadat & Harrison 2000, Marks et al. 2001). An important difference in the catalytic mechanism is that whereas TIM catalyses the interconversion between DHAP and DGAP, and an important aspect is that the elimination of the phosphate has to be prevented, methylglyoxal synthase does not bind DGAP, and the elimination reaction is actually catalyzed (Tsai & Gracy 1976). The flexibility of the catalytic glutamate side chain is an essential feature in the catalytic mechanism of TIM, as seen in this study as well as others. Methylglyoxal synthase has a shorter Asp instead of Glu at an equivalent position in the active site, which implies that the loss of flexibility of this residue does not let the reaction to proceed to the formation of DGAP (Zhang et al. 2002). Recent computational studies suggest that the previously discussed stereochemical effects in TIM do not have a major role in preventing phosphate elimination. Instead, more likely explanations include preventing the reaction from proceeding, as mentioned above, and stabilizing of the leaving phosphate by protonation and hydrogen bonding interactions (Zhang et al. 2002). Considering these facts, the correct timing of the catalysis and loop motion would become even more important. 67

In the 1.2-Å resolution TIM-DHAP complex (Jogl et al. 2003), a short hydrogen bond is observed between the catalytic Glu side chain and both C1 and C2 atoms of DHAP. This is in agreement with the observed LBHB-like hydrogen bond in the TIM-PGA complex in this study. C-O hydrogen bonds are not common in Nature. In the TIM active site, there are many other groups that would be more preferable hydrogen-bonding partners for Glu167. Still, the Glu chooses this carbon atom for the proton abstraction. There must be several features in the enzyme environment that favour this. The position of the glutamate is very precise, and the mainly hydrophobic environment helps in bringing the pKa’s of the Glu and the C1 atom of the substrate closer to each other. The pKa of a free glutamic acid is near 4.5 (Qasim et al. 1995). It is estimated that the pKa of a buried Glu could be around 7, as is also seen for the L. mexicana TIM, which has a most likely protonated glutamate buried in the dimer interface (Williams et al. 1999). This is still very far from the pKa of the carbon atom in the substrate. The developing negative charge on the substrate is stabilized by the oxyanion holes, which are present to stabilize the intermediates in both directions of the reaction. If this stabilization is strong enough, it would enable the reaction to proceed even if the proton would be on the glutamate just for a minor fraction of time. The active site is at the dimer interface and residues from all of the TIM barrel b strands or the following loops contribute to the active-site framework. Apparently crucial stabilizing interactions are provided by residues from loop 3 of the opposing subunit. The active-site region displays generally very low anisotropy, except for the ligand and the catalytic Glu and His. It seems that a very rigid framework is necessary to allow just the necessary amount and direction of flexibility for the key catalytic components that have to move to be able to shuttle protons.

6.3.3 Loop 6 and loop 7

Loop 6 is a flexible loop, which is known to be in an open conformation in the unliganded state and in a closed conformation in the liganded state. The conformational switch concerns a motion of about 7 Å for the tip of the loop. The exact timing of the loop motion and the synchronization with the catalytic events must be essential for TIM to work in an efficient way. It is fascinating how the enzyme is able to link together the loop motion and the chemistry in the active site. Loop 6 can be divided into an N-terminal hinge (Pro168, Val169, and Trp170), a rigid tip (Ala171, Ile172, Gly173, Thr174, and Gly175), and a C- terminal hinge (Lys176, Val177, and Ala178) (Sun & Sampson 1999, Xiang et al. 2001). After this, helix 6 starts with Thr179 as the N-cap residue, followed by Pro180 in the N-cap+1 position. The tip of the loop moves as a rigid body, while small changes in the f and j angles of the N- and C-terminal hinge cause loop opening and closure. The largest changes occur for j (Lys176) and f (Val177), which are +57° and -38°, respectively (Wierenga et al. 1991b). In the closed conformation, as seen in the atomic-resolution structure, the Ile172 side chain 68 interacts with the triose moiety of the substrate, as well as with the Glu167 side chain, whereas N of Gly173 is part of the phosphate-binding pocket. In the closed conformation, loop 6 tightly interacts via O of Ala171 with Og of Ser213 (in loop 7) and via N of Gly173 with the phosphate of PGA. Weaker hydrogen bonding is seen for the C-terminal part, between N of Gly175 and Og of Ser213 and between N of Ala178 and Oh of Tyr210; also the main chain of this part of the loop shows one of the highest anisotropy properties of the entire structure. It seems therefore that the C-terminal part of loop 6 has evolved not to interact too tightly with loop 7, allowing for efficient loop opening, which is a requirement for efficient catalysis (Rozovsky & McDermott 2001, Rozovsky et al. 2001). These structural features are in good agreement with NMR and computational studies. For example, solid-state NMR studies (Williams & McDermott 1995) have shown that the opening of loop 6 is not ligand-gated; instead, also in the liganded state, the loop opens at a rate in the millisecond timescale. Computational studies have also indicated that loop opening is initiated by the loosening of the hydrogen- bonding interactions between the C-terminal region of loop 6 and the YGGS motif of loop 7 (Joseph & Petsko 1990, Derreumaux & Schlick 1998). The observed interactions between loop 6 and the YGGS motif of loop 7 require small structural rearrangements of the Tyr210 and Ser213 side chains as compared to the open conformation (Noble et al. 1993). These rearrangements are correlated with peptide rotations of the peptide bonds after Gly211 and Gly212, which generate the phosphate-binding pocket as well as are required for the Glu167 side chain to move into the ”swung-in” position. As seen from the atomic-resolution structure, the pyrrolidine ring of Pro168 in the N-terminal region of loop 6 is planar. This is unusual, as the proline ring is known to occur in either of two different conformational states; ”up” (c1=-25°) or ”down” (c1=+25°) (Nemethy et al. 1992, Vitagliano et al. 2001) which differ in the position of the Cg atom with respect to the other members of the 5-atom ring. The properties of the pyrrolidine ring of proline confer a unique structural role for this residue (MacArthur & Thornton 1991). In the TIM-PGA structure in this study, both puckerings of proline rings are observed, except for Pro168, which has c1 of -2°. This conformation is enforced by close van der Waals contacts of the Cg atom with the side chains of Tyr166 and Ala171. This strained conformation is undoubtedly an important structural feature of the closed form of loop 6. For example, in natural TIM sequences, this proline is fully conserved, and random mutagenesis studies have shown that active variants require a proline at this position (Xiang et al. 2001). This strained conformation, not seen in the open loop 6, will destabilize the closed loop, thus facilitating loop opening and product release at the end of the reaction cycle. In the closed form of loop 6, the side chain of Ile172 folds over the substrate, shielding it and the catalytic glutamate from bulk solvent. Glu167 and the substrate are also contacted by Leu232. In this way, the Glu167 side chain in the closed conformation is buried in a pocket formed by the side chains of Ile172 and Leu232, but also lined by Ca of Gly211, O of Leu232, Sg of Cys126, and Wat28, all highly conserved residues. This pocket does not anchor the glutamate side chain via specific hydrogen bonds to the rest of the protein, and the width of the 69 pocket (the distance from Cd1 of Ile172 to Cg of Leu232 is 8.35 Å) allows for some conformational flexibility for this side chain. This conformational flexibility correlates with the proton shuttling function of Glu167. The importance of this hydrophobic pocket is highlighted by the full sequence conservation of Leu232 and Ile172. Moreover, a disease related point mutation Ile172Val has been characterized (Arya et al. 1997). The fact that the lack of merely one methyl group in the vicinity of the catalytic Glu side chain has such dramatic consequences highlights the precision of the enzyme to position the catalytic residues and allow them just the right amount of flexibility. 7 Concluding remarks

The initial questions in the beginning of this study concerned the importance of the fully conserved Arg191-Asp227 salt bridge far from the active site and the dimer interface, as well as properties of the flexible loop 6 - especially of the hinge regions. As always in research, answers to some of the questions were found, but many new ones were raised on the way. Surprisingly, the salt bridge does not seem to be of great importance for the catalytical properties of TIM, but instead, is a rather crucial factor for the efficient folding of the protein. When the unique properties of the L. mexicana E65Q crystals were discovered, the focus of this work was directed more towards the mechanistic aspects of the reaction catalyzed by TIM. Computational studies had already pointed out the possibility of Asn11 playing a role in stabilizing a reaction intermediate. This was confirmed by the crystal structure in this study, where strong hydrogen bonding was detected between this residue and the new aldehyde transition- state analogue. For the first time, it was also seen that the ketone and aldehyde substrate actually bind in a different way in the active site, which in turn affects the positioning of the catalytic Glu167 side chain. The atomic-resolution structure of the TIM-PGA complex highlights the mobility of the ligand and the Glu167 sidechain. Furthermore, a very short hydrogen bond is observed between the carboxylate oxygens of PGA and the glutamate. The length of this hydrogen bond is comparable to LBHBs, the role of which in enzyme catalysis has been vividly discussed by both experimentalists and theoreticians during the past years. The atomic-resolution structure also gives new insights into the factors regulating the timing of the opening and closure of the flexible loop 6. It is seen that the C-terminal part of the loop interacts with loop 7, but these interactions are rather loose, as the main chain at this region is seen in two distinct conformations in the liganded structure. The anisotropy of this region is also among the highest in the whole structure, and has a direction equivalent to the loop opening/closure event. In the N-terminus of the loop, there is a highly unusual planar proline side chain. This is expected to cause strain in the closed loop and favour loop opening. The work concerning the properties of the hinge regions of loop 6 is still on- going and will be finished in the near future. For sure, the results reported in 71 this thesis will also give new insights into the interpretation of the results obtained from the loop 6 work. The refinement of the atomic-resolution structures of TIM-PGH and TIM-IPP complexes has been initiated. These structures will enable comparisons of the binding modes of these two transition- state analogues from the ketone and aldehyde side of the reaction with very high precision. NMR studies have indicated the presence of an LBHB between the N- OH group of PGH and the catalytic Glu167 (Harris et al. 1997); it will be interesting to see the precise geometry of the Glu-PGH interaction in the crystal structure . IPP is the only aldehyde transition-state resembling compound, and as such, of high interest, as there currently is only the 2-Å resolution structure from this study available. It is hoped that the results of the present work, together with these future studies, will be able to establish the relative importance of the alternative mechanisms of the proton transfer steps. References

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