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A Decade of Protein Engineering on Ribonuclease T1

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A Decade of Protein Engineering on Ribonuclease T1 Vrije Universiteit Brussel A decade of protein engineering on ribonuclease T-1 - Atomic dissection of the enzyme- substrate interactions Steyaert, J Published in: European Journal of Biochemistry DOI: 10.1111/j.1432-1033.1997.t01-1-00001.x Publication date: 1997 Document Version: Final published version Link to publication Citation for published version (APA): Steyaert, J. (1997). A decade of protein engineering on ribonuclease T-1 - Atomic dissection of the enzyme- substrate interactions. European Journal of Biochemistry, 247(1), 1-11. https://doi.org/10.1111/j.1432- 1033.1997.t01-1-00001.x General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Eur. J. Biochem. 247, 1 - 11 (1997) 0 FEBS 1997 A decade of protein engineering on ribonuclease TI Atomic dissectiop of the enzyme-substrate interactions Jan STEYAERT Dienst Ultrastruktuur, Vlaams Interuniversitair instituut Biotechnologie, Vrije Universiteit Brussel, Belgium (Received 13 January 1997) - EJB 97 035110 During the last decade, protein engineering has been used to identify the residues that contribute to the ribonulease-TI-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2’-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the %‘-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and PhelOO further stabilize the transition state of the reaction. A functionally independent subsite, including Am36 and Asn98, contrib- utes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease TI guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base. Keywords: ribonuclease T, ; protein engineering; catalytic mechanism ; functional cooperativity ; substrate specificity ; subsite interaction ; nucleotide binding ; double-mutant cycle. Ribonuclease T, (RNase TI) of the slime mould Aspergillus (Fig. 2), yielding a 2’,3’-cyclophosphate [I, 23, 241. In a second, oryzae [ 1-41 is the best known representative of a large family separate step, this cyclic product may be hydrolyzed to yield 3’- of homologues microbial ribonucleases with members in the guanylic acid [25 -271. The catalyzed transesterification reac- prokaryotic and the eukaryotic world [5, 61. These ribonucleases tion consists of an associative nucleophilic displacement at the span the greatest evolutionary divide of all known protein fami- phosphorus atom of the 5’ leaving group by the entering 2’- lies. RNase TI,a guanosine-specific ribonuclease, was first iso- oxygen atom. The enzyme is thought to follow a concerted in- lated by Sato and Egami [I] from an enzyme extract (Takadias- line mechanism with a trigonal bipyramidal transition state, im- tase) of the culture medium of A. oryzae used in the malting plying a base and an acid located on either side of the scissile process of sake brewing. The enzyme consists of a single polypeptide chain of 104 residues of known sequence [7, 81 and contains two disulfide bridges. RNase T1 is a stable acidic pro- tein; it is fairly resistant to heat, acids, and bases. A large number of crystal structures of RNase T, are available in dif- ferent liganded states [9-221. The general fold (Fig. 1) consists of a 4.5-turn a helix and two antiparallel p sheets, connected through a series of wide loops [9, 101. The residues implicated in catalysis are anchored in the major p sheet, while guanosine specificity is defined by residues in loop regions. RNase T, cleaves the P-05’ ester bond of GpN sequences of single-stranded RNA by a transphosphorylation reaction Correspondence to J. Steyaert, Dienst Ultrastruktuur, Vrije Universi- teit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium Fax: +32 2 359 02 89. E-mail: jstey [email protected]. be URL: http://imol.vub.ac.be Abbreviations. GpMe, guanosine 3’-(methyl phosphate) ; GpRib, guanosine 3’-(5’-~-ribosyl phosphate); NpN, 3’,5’-linked dinucleoside monophosphate compounds; RNase T,, ribonuclease T,. Fig. 1. Ribbon drawing showing the overall structure of RNase T,. Enzymes. Ribonuclease T, (EC 3.1.27.3); ribonuclease A (EC The competitive inhibitor 2’-GMP is shown in the ball-and-stick repre- 3.1.27.5); micrococcal nuclease (Ec 3.1.31.l). sentation. 2‘-GMP binds on top of the major p-pleated sheet. The guanine Note. This Review will be reprinted in EJB Reviews 1997 which will base makes extensive contacts with a loop that connects two /j stands. be available in April 1998. Coordinates were taken from [lo]. 2 Steyaert (Em J. Biochern. 247) 7 transphosphorylation hydrolysis R I 8 a Y70 OH I -o-p=o c?? O, lo cw I 0 I o,7 P\o. -0-P=o I 1 0' ROH cc?0 OH 04-0\ OR I b CH2 HE \/ P PO0 AH A: + RCH20H Fig. 2. The chemistry and stereochemistry of the RNase-TI-catalyzedreactions. (a) The sequence of events in the RNase-TI-catalyzeddepolymer- ization of RNA. G represents the guanine base. (b) The stereochemistry of the transphosphorylation reaction. A, acid, B, base. bond [27-301. RNase TI has a pronounced specificity for gua- Table 1. Dependence of the steady-state kinetic parameters of RNase nine; kinetic studies on the transesterification of dinucleoside T, on the nature of the leaving group of the substrate. Measurements phosphates revealed that the specificity constant (k,.,,/K,,,) for were performed in imidazole buffer (0.1 M ionic strength), pH 6.0, at 35°C [47, 481. The wild-type/GpC and wild-type/GpA reactions obey GpN substrates is about 10h-fold greater than that for corre- Briggs-Haldane kinetics (K,,, > Ks). Equilibrium dissociation constants sponding ApN substrates and at least 108-fold greater than that have been taken from Steyaert et al. [31]. for CpN and UpN substrates [24]. Kinetic measurements on RNase T1 indicate that the second-order rate constants for the transphosphorylation of the dinucleoside phosphate model sub- strates GpC, GpA, and polymeric yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site [31, 321. Further improvements in the efficiency GPC 29.5 216 348 1611 of the chemical transformation step would lead to only small GPA 56 78 81 1038 increases in the rate of turnover at low substrate concentrations GPU 33 33 29 879 GpRih 100 100 9.3 93 [33]. Since the RNA content of most natural environments may GpMe 52 52 0.03 0.577 be expected to be low, the extracellular RNase T, seems to have been perfected catalytically from an evolutionary point of view 1311. Enzymes catalyze reactions by binding to the transition state of RNase T, for the chemical transition state. The cloning and of the chemical interconversion tighter than to the substrate and expression of recombinant RNase-T, a decade ago [38-421 ren- product ground states [34, 351, i.e. catalysis is recognition [36]. dered this enzyme amenable to detailed structure-function analy- The rate enhancement mediated by an enzyme may thus be de- sis by means of protein-engineering techniques [36]. scribed in terms of a set of intermolecular binding interactions that positively discriminate the transition state relative to the subsite-binding energy for chemical turnover substrates and the products. The ability of an enzyme to bind The use of the transition state is given by the ratio of k,,jK,,, of the catalyzed Mapping of the subsite. It is well established that enzymes reaction and the uncatalyzed rate (k.,,,,,) [37]. The affinity of degrading polymeric substrates contain subsites for secondary RNase T, for the transition state of GpA transphosphorylation substrate units, the interaction being used mostly to increase k,,, has been estimated at 3 X 10- Is M [32].Because the rate-limiting rather than to bind the substrate [43]. For RNase TI,the turnover step of the RNase TI-catalyzed transphosphorylation of GpA is numbers (kJ for the trunsesterification of dinucleotide phos- not the chemical turnover but the association of enzyme and phate substrates follow the order GpC > GpA > GpU (Table substrate [31], this value is an upper limit for the dissociation l), whereas the equilibrium dissociation constants (K,) for the constant of the enzyme for the chemical transition state. This substrates are very similar [31, 44-46], indicating that RNase review focuses on the molecular basis of the remarkable affinity T, contains a subsite with a preference for cytosine that contrib- Steyaert (Em J. Biochem. 247) 3 transphosphorylation rate of GpC compared with those of GpRib and GpMe (Fig. 4). The dependence of these mutational effects on the presence of the leaving cytidine base or the leav- ing ribose indicate that the Am36 and Am98 amide functions are part of the subsite. It appears that Asn36 interacts with the ribose moiety of the leaving nucleoside.
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