DOCSLIB.ORG

Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition Inhibition of Enzyme Activity Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition Competitive Inhibition Fig 8-15 1 Competitive Inhibition COMPETITIVE Equilibria Scheme -Ic structrually resembles S, but is not an S E + S ES P + E K -Ic binds to free E at active site where S binds + m -I competes with S for free E c Ic -High S overcomes inhibition because all E K is bound in ES complex; since rate [ES] and c [ES] is max, rate is max; no EIc is present EI c slope = Km . (1+ [I c] / K c) / Vmax +I c -Ic 1 / Vo +Ic -I Vo y-int = 1/Vmax c So 1 / So x-int = -1/Km slope = Km/Vmax yint = -1 / (Km . (1+ [I c ] / Kc )) Noncompetitive Inhibition Fig 8-15 2 Noncompetitive Inhibition NONCOMPETITIVE Equilibria Scheme -Inc is not structurally similar to S; is not an S E + S ES P + E + Km -Inc binds to free E or ES at a site where S + does not bind I nc I nc -Inc does NOT compete with S for free E K nc K'nc -High S cannot overcome inhibition because Inc binds to ES complex, inactivating it EI nc + S ESI nc (inactive) - Inc slope = K m . (1+ [In c ] / Kc ) / Vmax +I + Inc 1 / V Vo o -I So 1 / So y-int = (1+[I nc] / Knc ) / Vmax slope = K m/Vmax x-int = -1/Km y-int = 1/Vmax Examples: Competitive and Noncompetitive Inhibition H+ O H O H H O OH C - C + CH C CO - H2N + CH3 C CO2 NH2 3 2 H N N pyruvate L-lactate R R **NADH (reduced form) **NAD (oxidized form) ORDERED BI BI MECHANISM + NADH PYR LAC NAD S1 S2 P1 P2 E ES1 ES1S2 EP1P2 EP2 E 3 Examples: Competitive and Noncompetitive Inhibition LACTATE DEHYDROGENASE H+ O H O H H O OH C - C + CH C CO - H2N + CH3 C CO2 NH2 3 2 H N N pyruvate L-lactate R R **NADH (reduced form) **NAD (oxidized form) HO H O O O C NH - - INHIBITORS: 2 H2N C CO2 CH3CH2HN C CO2 N oxamate ethyloxamate R NAD-OH For multisubstrate rxns, the type of inhibition depends upon the substrate that is varied in the inhibition experiment! LACTATE DEHYDROGENASE H+ O H O H H O OH C - C + CH C CO - H2N + CH3 C CO2 NH2 3 2 H N N pyruvate L-lactate R R **NADH (reduced form) **NAD (oxidized form) HO H O C What substrate does this INHIBITORS: NH2 inhibitor resemble? N R NAD-OH NADH 4 Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when NADH is the varied substrate? + H ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R O **NADH HO H E ES ES S EP P EP2 E (reduced form) **NAD 1 1 2 1 2 C (oxidized form) NH2 N Competitive inhibition R NAD-OH seen if varied S is NADH Noncompetitive Competitive +I +I 1 / Vo 1 / Vo -I -I 1/NADH 1/NADH Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when pyruvate is the varied substrate? + H ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R O **NADH HO H E ES ES S EP P EP2 E (reduced form) **NAD 1 1 2 1 2 C (oxidized form) NH2 N Noncompetitive inhibition R NAD-OH seen if varied S is pyruvate Noncompetitive Competitive +I +I 1 / Vo 1 / Vo -I -I 1/PYRUVATE 1/PYRUVATE 5 Which plot describes the inhibition of lactate dehydrogenase by oxamate when NADH is the varied substrate? + H ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R **NADH O EP **NAD E ES1 ES1S2 EP1P2 2 E (reduced form) - (oxidized form) H2N C CO2 oxamate Noncompetitive inhibition seen if varied S is NADH Noncompetitive Competitive +I +I 1 / Vo 1 / Vo -I -I 1/NADH 1/NADH Which plot describes the inhibition of lactate dehydrogenase by oxamate when pyruvate is the varied substrate? + H ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R **NADH O EP **NAD E ES1 ES1S2 EP1P2 2 E (reduced form) - (oxidized form) H2N C CO2 oxamate Competitive inhibition seen if varied S is pyruvate Noncompetitive Competitive +I +I 1 / Vo 1 / Vo -I -I 1/PYRUVATE 1/PYRUVATE 6 Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind UNCOMPETITIVE Equilibria Scheme -Iu is not structurally similar to S; is not an S E + S ES P + E Km -Iu binds to ES only; S opens up a site four I + -Iu binding site may be in active site but binding Iu of I u requires prior binding of S K -High S cannot overcome inhibition because presence u u of S is required to provide a site for binding of I EIu y-int = (1+ [Iu] / Ku ) / V max + Iu +I 1/Vo - Iu -I Vo slope = Km / Vmax So 1 / So x-int = -(1+ [Iu] / Ku ) / Km y-int = 1 / Vmax x-int = -1 / Km 7 Example: Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE H O O O O H H O C C H O - NH C O P O 2 + - C - H C OH + HO P O H2N O + H C OH N OH CH2OPi N CH2OPi R glyceraldehyde-3-Pi R 1,3-bisphosphoglycerate **NAD **NADH (oxid ized fo rm ) (red uce d fo rm) O INHIBITOR: HO As O- ORDERED TRI BI MECHANISM OH NAD + G AP Pi 1,3- BPG N ADH S1 S2 S3 P1 P2 E ES1 ES'1P2 ES'1P2 E EP1P2 EP2 . - Predict the type of inhibition by H2AsO4 when each of the substrates is varied in inhibition experiments This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE H O O O O H H O C C H O - NH C O P O 2 + - C - H C OH + HO P O H2N O + H C OH N OH CH2OPi N CH2OPi R glyceraldehyde-3-Pi R 1,3-bisphosphoglycerate **NAD **NADH (oxid ized fo rm ) (red uce d fo rm) O INHIBITOR: HO As O- ORDERED TRI BI MECHANISM OH NAD + G AP Pi 1,3- BPG N ADH S1 S2 S3 P1 P2 E ES1 ES'1P2 ES'1P2 E EP1P2 EP2 . 8 - Predict the type of inhibition by H2AsO4 when each of the substrates is varied in inhibition experiments ORDERED TRI BI MECHANISM O + GAP Pi O 1,3-BPG NADH NAD HO AsO- HO P O- P P S1 S2 S3 1 2 OH OH ES'1P2 ES' P E E ES1 1 2 EP1P2 EP2 . Noncompetitive Competitive Uncompetitive +I +I 1 / Vo 1 / Vo 1 / Vo +I -I -I -I 1/So 1/S o 1/S o If So = Pi If So = NADH or GAP Product Inhibition Equilibria Scheme PRODUCT INHIBITION E + S ES P + E K -Ip is structurally similar to S + m -Ip binds to free E at active site where S binds P -Ip competes with S for free E -At low S, resembles competitive inhibition Kp -However, at high S, the inhibition is not overcome EI because higher levels of P are generated which p inhibit the enzyme 1 / Vo Vo slope = Km / Vmax So 1 / So x-int = -1 / Km 9 Example: Product Inhibition b-galactosidase (lactase) HOCH lactose 2 O HOCH2 OH OH HO O O OH OH O OH H H HOCH2 HOCH2 HO O OH O OH OH HO OH OH OH galactose glucose Suicide Inhibition This type of enzyme inhibition results in the stoichiometric covalent modification of a side chain on an amino acid in the active site of an enzyme. The inhibitor chemically resembles a (one of the) substrate(s) and binds in the active site in the same way as the substrate(s) binds. The inhibitor, however, has a functional group, ususally a leaving group, that is replaced by a nucleophile in the enzyme active site. This covalent enzyme-inhibitor complex forms irreversibly, thereby irreversibly inactivating the enzyme. Therefore this type of inhibition is called "suicide inhibition" or affinity labeling and the inhibitor is called a "suicide inhibitor". This reaction with the suicide inhibitor removes active enzyme from the system; this removal is measured as inhibition. Since active enzyme is lost, the inhibition is not relieved at high substrate levels. The rate, at high substrate in the presence of the inhibitor,is still proportional to the amount of the enzyme-substrate complex.
Load more

Recommended publications
  • Allosteric Regulation in Drug Design
    Mini Review Curr Trends Biomedical Eng & Biosci Volume 4 Issue 1 - May 2017 Copyright © All rights are reserved by Ashfaq Ur Rehman DOI: 10.19080/CTBEB.2017.04.5555630 Allosteric regulation in drug design Ashfaq Ur Rehman1,2*, Shah Saud3, Nasir Ahmad4, Abdul Wadood2 and R Hamid5 1State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, China 2Department of Biochemistry, Abdul Wali Khan University Mardan, Pakistan 3Laboratory of Analytical Biochemistry and Bio separation, Shanghai Jiao Tong University, China 4Department of Chemistry, Islama College University Peshawar, Pakistan 5Department of Bioinformatics, Muhammad Ali Jinnah University Islamabad, Pakistan Submission: May 02, 2017; Published: May 23, 2017 *Corresponding author: Ashfaq Ur Rehman, State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China, Tel: ; Fax: 86-21-34204348; Email: Abstract mechanism, which are initiated through attachment of ligand or inhibitors with the protein or enzymes other than active (orthosteric) sites. ThisProtein mini review and enzymes involved play mechanism, significant types roles and in importancebiological processes of allosteric of all regulations living organisms; in drug theirdesign functions process. are regulated through allosteric Keywords: Allosteric, Activator: Drug design Introduction and ultimately cause disease. While various biological processes expressed the control at different points in life time of protein function is pivotal. As all the cell processes are under carful For the survival of all organisms the significance of protein included regulation of gene expression, translation into protein control and if not properly controls this leads to the abnormality through control of activity and at last degradation of protein [1].
    [Show full text]
  • Tyrosine Kinase – Role and Significance in Cancer
    Int. J. Med. Sci. 2004 1(2): 101-115 101 International Journal of Medical Sciences ISSN 1449-1907 www.medsci.org 2004 1(2):101-115 ©2004 Ivyspring International Publisher. All rights reserved Review Tyrosine kinase – Role and significance in Cancer Received: 2004.3.30 Accepted: 2004.5.15 Manash K. Paul and Anup K. Mukhopadhyay Published:2004.6.01 Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S Nagar, Mohali, Punjab, India-160062 Abstract Tyrosine kinases are important mediators of the signaling cascade, determining key roles in diverse biological processes like growth, differentiation, metabolism and apoptosis in response to external and internal stimuli. Recent advances have implicated the role of tyrosine kinases in the pathophysiology of cancer. Though their activity is tightly regulated in normal cells, they may acquire transforming functions due to mutation(s), overexpression and autocrine paracrine stimulation, leading to malignancy. Constitutive oncogenic activation in cancer cells can be blocked by selective tyrosine kinase inhibitors and thus considered as a promising approach for innovative genome based therapeutics. The modes of oncogenic activation and the different approaches for tyrosine kinase inhibition, like small molecule inhibitors, monoclonal antibodies, heat shock proteins, immunoconjugates, antisense and peptide drugs are reviewed in light of the important molecules. As angiogenesis is a major event in cancer growth and proliferation, tyrosine kinase inhibitors as a target for anti-angiogenesis can be aptly applied as a new mode of cancer therapy. The review concludes with a discussion on the application of modern techniques and knowledge of the kinome as means to gear up the tyrosine kinase drug discovery process.
    [Show full text]
  • DNA Breakpoint Assay Reveals a Majority of Gross Duplications Occur in Tandem Reducing VUS Classifications in Breast Cancer Predisposition Genes
    © American College of Medical Genetics and Genomics ARTICLE Corrected: Correction DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes Marcy E. Richardson, PhD1, Hansook Chong, PhD1, Wenbo Mu, MS1, Blair R. Conner, MS1, Vickie Hsuan, MS1, Sara Willett, MS1, Stephanie Lam, MS1, Pei Tsai, CGMBS, MB (ASCP)1, Tina Pesaran, MS, CGC1, Adam C. Chamberlin, PhD1, Min-Sun Park, PhD1, Phillip Gray, PhD1, Rachid Karam, MD, PhD1 and Aaron Elliott, PhD1 Purpose: Gross duplications are ambiguous in terms of clinical cohort, while the remainder have unknown tandem status. Among interpretation due to the limitations of the detection methods that the tandem gross duplications that were eligible for reclassification, cannot infer their context, namely, whether they occur in tandem or 95% of them were upgraded to pathogenic. are duplicated and inserted elsewhere in the genome. We Conclusion: DBA is a novel, high-throughput, NGS-based method investigated the proportion of gross duplications occurring in that informs the tandem status, and thereby the classification of, tandem in breast cancer predisposition genes with the intent of gross duplications. This method revealed that most gross duplica- informing their classifications. tions in the investigated genes occurred in tandem and resulted in a Methods: The DNA breakpoint assay (DBA) is a custom, paired- pathogenic classification, which helps to secure the necessary end, next-generation sequencing (NGS) method designed to treatment options for their carriers. capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. Genetics in Medicine (2019) 21:683–693; https://doi.org/10.1038/s41436- Results: DBA allowed us to ascertain breakpoints for 44 unique 018-0092-7 gross duplications from 147 probands.
    [Show full text]
  • Enzyme Activity and Assays Introductory Article
    Enzyme Activity and Assays Introductory article Robert K Scopes, La Trobe University, Bundoora, Victoria, Australia Article Contents . Factors that Affect Enzymatic Analysis Enzyme activity refers to the general catalytic properties of an enzyme, and enzyme assays . Initial Rates and Steady State Turnover are standardized procedures for measuring the amounts of specific enzymes in a sample. Measurement of Enzyme Activity . Measurement of Protein Concentration Factors that Affect Enzymatic Analysis . Methods for Purifying Enzymes . Summary Enzyme activity is measured in vitro under conditions that often do not closely resemble those in vivo. The objective of measuring enzyme activity is normally to determine the exactly what the concentration is (some preparations of amount of enzyme present under defined conditions, so unusual substrates may be impure, or the exact amount that activity can be compared between one sample and present may not be known). This is because the rate another, and between one laboratory and another. The measured varies with substrate concentration more rapidly conditions chosen are usually at the optimum pH, as the substrate concentration decreases, as can be seen in ‘saturating’ substrate concentrations, and at a temperature Figure 1. In most cases an enzyme assay has already been that is convenient to control. In many cases the activity is established, and the substrate concentration, buffers and measured in the opposite direction to that of the enzyme’s other parameters used previously should be used again. natural function. Nevertheless, with a complete study of There are many enzymes which do not obey the simple the parameters that affect enzyme activity it should be Michaelis–Menten formula.
    [Show full text]
  • Revised Glossary for AQA GCSE Biology Student Book
    Biology Glossary amino acids small molecules from which proteins are A built abiotic factor physical or non-living conditions amylase a digestive enzyme (carbohydrase) that that affect the distribution of a population in an breaks down starch ecosystem, such as light, temperature, soil pH anaerobic respiration respiration without using absorption the process by which soluble products oxygen of digestion move into the blood from the small intestine antibacterial chemicals chemicals produced by plants as a defence mechanism; the amount abstinence method of contraception whereby the produced will increase if the plant is under attack couple refrains from intercourse, particularly when an egg might be in the oviduct antibiotic e.g. penicillin; medicines that work inside the body to kill bacterial pathogens accommodation ability of the eyes to change focus antibody protein normally present in the body acid rain rain water which is made more acidic by or produced in response to an antigen, which it pollutant gases neutralises, thus producing an immune response active site the place on an enzyme where the antimicrobial resistance (AMR) an increasing substrate molecule binds problem in the twenty-first century whereby active transport in active transport, cells use energy bacteria have evolved to develop resistance against to transport substances through cell membranes antibiotics due to their overuse against a concentration gradient antiretroviral drugs drugs used to treat HIV adaptation features that organisms have to help infections; they
    [Show full text]
  • The Self-Inhibitory Nature of Metabolic Networks and Its Alleviation Through Compartmentalization
    ARTICLE Received 30 Oct 2016 | Accepted 23 May 2017 | Published 10 Jul 2017 DOI: 10.1038/ncomms16018 OPEN The self-inhibitory nature of metabolic networks and its alleviation through compartmentalization Mohammad Tauqeer Alam1,2, Viridiana Olin-Sandoval1,3, Anna Stincone1,w, Markus A. Keller1,4, Aleksej Zelezniak1,5,6, Ben F. Luisi1 & Markus Ralser1,5 Metabolites can inhibit the enzymes that generate them. To explore the general nature of metabolic self-inhibition, we surveyed enzymological data accrued from a century of experimentation and generated a genome-scale enzyme-inhibition network. Enzyme inhibition is often driven by essential metabolites, affects the majority of biochemical processes, and is executed by a structured network whose topological organization is reflecting chemical similarities that exist between metabolites. Most inhibitory interactions are competitive, emerge in the close neighbourhood of the inhibited enzymes, and result from structural similarities between substrate and inhibitors. Structural constraints also explain one-third of allosteric inhibitors, a finding rationalized by crystallographic analysis of allosterically inhibited L-lactate dehydrogenase. Our findings suggest that the primary cause of metabolic enzyme inhibition is not the evolution of regulatory metabolite–enzyme interactions, but a finite structural diversity prevalent within the metabolome. In eukaryotes, compartmentalization minimizes inevitable enzyme inhibition and alleviates constraints that self-inhibition places on metabolism. 1 Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK. 2 Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK. 3 Department of Food Science and Technology, Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubira´n, Vasco de Quiroga 15, Tlalpan, 14080 Mexico City, Mexico.
    [Show full text]
  • Opportunities for Catalysis in the 21St Century
    Opportunities for Catalysis in The 21st Century A Report from the Basic Energy Sciences Advisory Committee BASIC ENERGY SCIENCES ADVISORY COMMITTEE SUBPANEL WORKSHOP REPORT Opportunities for Catalysis in the 21st Century May 14-16, 2002 Workshop Chair Professor J. M. White University of Texas Writing Group Chair Professor John Bercaw California Institute of Technology This page is intentionally left blank. Contents Executive Summary........................................................................................... v A Grand Challenge....................................................................................................... v The Present Opportunity .............................................................................................. v The Importance of Catalysis Science to DOE.............................................................. vi A Recommendation for Increased Federal Investment in Catalysis Research............. vi I. Introduction................................................................................................ 1 A. Background, Structure, and Organization of the Workshop .................................. 1 B. Recent Advances in Experimental and Theoretical Methods ................................ 1 C. The Grand Challenge ............................................................................................. 2 D. Enabling Approaches for Progress in Catalysis ..................................................... 3 E. Consensus Observations and Recommendations..................................................
    [Show full text]
  • NADPH-Dependent A-Oxidation of Unsaturated Fatty Acids With
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6673-6677, August 1992 Biochemistry NADPH-dependent a-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms (5-enoyl-CoA/A3,A2-enoyl-CoA isomerase/A3',52'4-dienoyl-CoA isomerase/2,4-dienoyl-CoA reductase) TOR E. SMELAND*, MOHAMED NADA*, DEAN CUEBASt, AND HORST SCHULZ* *Department of Chemistry, City College of the City University of New York, New York, NY 10031; and tJoined Departments of Chemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471 Communicated by Salih J. Wakil, April 13, 1992 ABSTRACT The mitochondrial metabolism of 5-enoyl- a recent observation of Tserng and Jin (5) who reported that CoAs, which are formed during the (3-oxidation of unsaturated the mitochondrial -oxidation of 5-enoyl-CoAs is dependent fatty acids with double bonds extending from odd-numbered on NADPH. Their analysis of metabolites by gas chroma- carbon atoms, was studied with mitochondrial extracts and tography/mass spectrometry led them to propose that the purified enzymes of (3-oxidation. Metabolites were identified double bond of 5-enoyl-CoAs is reduced by NADPH to yield spectrophotometrically and by high performance liquid chro- the corresponding saturated fatty acyl-CoAs, which are then matography. 5-cis-Octenoyl-CoA, a putative metabolite of further degraded by P-oxidation. linolenic acid, was efficiently dehydrogenated by medium- This report addresses the question of how 5-enoyl-CoAs chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis- are chain-shortened by P-oxidation. We demonstrate that octadienoyl-CoA, which was isomerized to 3,5-octadienoyl- 5-enoyl-CoAs, after dehydrogenation to 2,5-dienoyl-CoAs, CoA either by mitochondrial A3,A2-enoyl-CoA isomerase (EC can be isomerized to 2,4-dienoyl-CoAs, which are reduced by 5.3.3.8) or by peroxisomal trifunctional enzyme.
    [Show full text]
  • On the Active Site Thiol of Y-Glutamylcysteine Synthetase
    Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2464-2468, April 1988 Biochemistry On the active site thiol of y-glutamylcysteine synthetase: Relationships to catalysis, inhibition, and regulation (glutathione/cystamine/Escherichia coli/kidney/enzyme inactivation) CHIN-SHIou HUANG, WILLIAM R. MOORE, AND ALTON MEISTER Cornell University Medical College, Department of Biochemistry, 1300 York Avenue, New York, NY 10021 Contributed by Alton Meister, December 4, 1987 ABSTRACT y-Glutamylcysteine synthetase (glutamate- dithiothreitol, suggesting that cystamine forms a mixed cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia disulfide between cysteamine and an enzyme thiol (15). coli strain enriched in the gene for this enzyme by recombinant Inactivation of the enzyme by the L- and D-isomers of DNA techniques. The purified enzyme has a specific activity of 3-amino-1-chloro-2-pentanone, as well as that by cystamine, 1860 units/mg and a molecular weight of 56,000. Comparison is prevented by L-glutamate (14). Treatment of the enzyme of the E. coli enzyme with the well-characterized rat kidney with cystamine prevents its interaction with the sulfoxi- enzyme showed that these enzymes have similar catalytic prop- mines. Titration of the enzyme with 5,5'-dithiobis(2- erties (apparent Km values, substrate specificities, turnover nitrobenzoate) reveals that the enzyme has a single exposed numbers). Both enzymes are feedback-inhibited by glutathione thiol that reacts with this reagent without affecting activity but not by y-glutamyl-a-aminobutyrylglycine; the data indicate (16). 5,5'-Dithiobis(2-nitrobenzoate) does not interact with that glutathione binds not only at the glutamate binding site but the thiol that reacts with cystamine.
    [Show full text]
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [Show full text]
  • Crystallography Captures Catalytic Steps in Human Methionine Adenosyltransferase Enzymes
    Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes Ben Murraya,b, Svetlana V. Antonyuka, Alberto Marinab, Shelly C. Luc, Jose M. Matod, S. Samar Hasnaina,1, and Adriana L. Rojasb,1 aMolecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZX, England; bStructural Biology Unit, Center for Cooperative Research in Biosciences, 48160 Derio, Spain; cDivision of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, CA 90048; and dCIC bioGUNE, CIBERehd, Parque Tecnologico Bizkaia, 801A-1.48160 Derio, Spain Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved January 8, 2016 (received for review June 4, 2015) The principal methyl donor of the cell, S-adenosylmethionine catalytic mechanism (13, 14) in which the reaction is initiated (SAMe), is produced by the highly conserved family of methionine through a nucleophilic attack by the sulfur atom of methionine adenosyltranferases (MATs) via an ATP-driven process. These en- on the C5′ atom of ATP, which produces the intermediate tri- zymes play an important role in the preservation of life, and their polyphosphate (PPPi). Hydrolysis of the PPPi into pyrophos- dysregulation has been tightly linked to liver and colon cancers. We phate (PPi) and orthophosphate (Pi) then occurs (Fig. S1). present crystal structures of human MATα2 containing various bound A common feature of MAT enzymes is a gating loop that α – ligands, providing a “structural movie” of the catalytic steps. High- to flanks the active site (in human MAT 2 residues 113 131), atomic-resolution structures reveal the structural elements of the which has been postulated to act in a dynamic way to allow ac- enzyme involved in utilization of the substrates methionine and cess to the active site.
    [Show full text]
  • Plant Protease Inhibitors: a Defense Strategy in Plants
    Biotechnology and Molecular Biology Review Vol. 2 (3), pp. 068-085, August 2007 Available online at http://www.academicjournals.org/BMBR ISSN 1538-2273 © 2007 Academic Journals Standard Review Plant protease inhibitors: a defense strategy in plants Huma Habib and Khalid Majid Fazili* Department of Biotechnology, The University of Kashmir, P/O Naseembagh, Hazratbal, Srinagar -190006, Jammu and Kashmir, India. Accepted 7 July, 2007 Proteases, though essentially indispensable to the maintenance and survival of their host organisms, can be potentially damaging when overexpressed or present in higher concentrations, and their activities need to be correctly regulated. An important means of regulation involves modulation of their activities through interaction with substances, mostly proteins, called protease inhibitors. Some insects and many of the phytopathogenic microorganisms secrete extracellular enzymes and, in particular, enzymes causing proteolytic digestion of proteins, which play important roles in pathogenesis. Plants, however, have also developed mechanisms to fight these pathogenic organisms. One important line of defense that plants have to fight these pathogens is through various inhibitors that act against these proteolytic enzymes. These inhibitors are thus active in endogenous as well as exogenous defense systems. Protease inhibitors active against different mechanistic classes of proteases have been classified into different families on the basis of significant sequence similarities and structural relationships. Specific protease inhibitors are currently being overexpressed in certain transgenic plants to protect them against invaders. The current knowledge about plant protease inhibitors, their structure and their role in plant defense is briefly reviewed. Key words: Proteases, enzymes, protease inhibitors, serpins, cystatins, pathogens, defense. Table of content 1.
    [Show full text]