DOCSLIB.ORG

Enzymes and Metabolic Pathways

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enzymes and Metabolic Pathways Enzymes & Metabolism Enzyme characteristics • Made of protein Enzymes and • Catalysts: – reactions occur 1,000,000 times faster with Metabolic Pathways enzymes – Not part of reaction • Not changed or affected by reaction • Used over and over Mechanism of Enzyme Action Why do cells need enzymes? • Ability of enzymes to lower activation energy • Not enough time to leave things to chance due to structure. • Most probable reactions are not the required • Each type of enzyme has has a highly-ordered, • Important molecules do not always occur in large characteristic 3-dimensional shape quantities (conformation). – Ridges, grooves, and pockets lined with specific amino acids. But since life processes are dependent upon enzymes, – Pockets active in catalyzing a reaction are called organisms cannot survive at high temperature the active sites of the enzyme. – Heat denatures proteins Reactants must collide for reaction Mechanism of Enzyme Action (continued) to occur • Substrates have specific shapes to fit into the active sites (lock- and-key model): – Substrate fits into active sites in enzyme. – Perfect fit may be induced: • Enzyme undergoes structural change. – Enzyme-substrate complex formed, then dissociates. – Products formed and enzyme is unaltered. Heyer 1 Enzymes & Metabolism Without enzymes, collisions are An enzyme brings reactants random together by binding to them Induced Fit Model Induced Fit Model 1. Enzyme binds 2. Substrates orient substrate, forms for productive 3. Productive collision 4. Reaction occurs, complex collision produces transition products are state released Naming of Enzymes Enzymes as Catalysts • Enzyme name ends with suffix “-ase.” • Name = substrate – action – “-ase” – E.g., glucose phosphorylase is an enzyme that adds a phosphate to glucose. – If the “action” is left out of the name, assume the action is hydrolysis. E.g., a protease catalyzes the hydrolysis of proteins into oligopeptides or amino acids. • Different organs may make different enzymes (isoenzymes) that have the same activity. – Differences in structure do not affect the active sites. Heyer 2 Enzymes & Metabolism Regulation of Enzyme Activity Reversible Reactions • Rate of enzyme-catalyzed reactions measured by the rate that • Some enzymatic reactions are reversible. substrates (reactants) are converted to products. – Both forward and backward reactions are catalyzed by • Factors regulating enzyme activity: same enzyme. 1. Mass action: concentration of substrates and products. carbonic anhydrase 2. Concentration of enzyme. • H 0 + C0 H C0 • Gene expression. 2 2 2 3 • Post-translational modification. • Proteolysis. • Law of mass action: 3. Alteration of enzyme structure. – Principal that reversible reactions will be driven from the • Activation. side of the equation where concentration is higher to side • Temperature. where concentration is lower. • pH. • But most biological reactions have complex organic 4. Cofactors and Coenzymes. substrates which require separate enzymes to catalyze 5. Inhibitors. the reverse reaction. Enzyme Activation Substrate Concentration • Enzymes may be produced in an inactive form. • Final post-translational modification occurs only • At a specific [enzyme], when enzyme is needed. rate of product formation increases as the [substrate] – In pancreas, digestive enzymes are produced as increases. inactive zymogens, which are activated in lumen of • Plateau of maximum intestine. velocity occurs when enzyme is saturated. • Part of the polypeptide is hydrolyzed off. • Additional [substrate] • Protects against self-digestion. does not not increase – In liver cells, enzyme is inactive when produced and reaction rate. is activated by addition of phosphate group. • Phosphorylation/dephosphorylation: Activation/inactivation of an enzyme. Enzyme Shape Effect of Temperature • Function of the enzyme is dependent on its shape • Rate of reaction increases as temperature increases. • Destruction of shape destroys function • Reaction rate plateaus, slightly above body temperature (37o C). – Heat • Reaction rate decreases as temperature increases. – Acid (H+) • Enzymes denature at high temperatures. Optimal temperature for Optimal temperature for typical human enzyme enzyme of thermophilic (heat-tolerant) bacteria Rate of reaction 0 20 40 80 100 Temperature (Cº) (a) Optimal temperature for two enzymes Functional shape Denatured Figure 8.18 Heyer 3 Enzymes & Metabolism Enzyme helpers: Cofactors Effect of pH • Each enzyme exhibits peak activity at narrow pH range (pH optimum). • pH optimum reflects the pH of the body fluid in which the enzyme is found. • If pH changed, so is no longer within the enzyme range; reaction will decrease. • Alter shape or charge polarity of enzyme: makes active site available to substrate • Metal ions from dietary minerals. Substrate Active site Enzyme Inhibitors Cofactors and Coenzymes Enzyme • Needed for the activity of specific • Competitive Inhibitors: enzymes. Competitive ß inhibitor • Cofactor: Shape at least partially fits – Attachment of cofactor causes a in the enzymes active site conformational change of active site. È blocks substrate from binding. – Participate in temporary bonds between enzyme and substrate. • Coenzymes: • Allosteric “different shape” Inhibitors: – Organic molecules derived from ß Bind to a different location H20 soluble vitamins. – Transport H+ and small molecules of the enzyme protein from one enzyme to another. — the allosteric site È alters the shape of the protein Allosteric inhibitor È active site no longer fits substrates Figure 8.19 Most enzymes have allosteric regulation Enzymes are Binding Proteins • Proteins change shape when regulatory molecules bind to sites. • I.e., they function by binding to a substrate (ligand) – Allosteric activators (cofactors) and/or inhibitors • Other important biological regulation by means of binding proteins: Allosteric activater stabilizes active from • Receptors for hormones & neurotransmitters Allosteric enyzme Active site with four subunits (one of four) • Transporters across cell membranes • Carriers of low-solubility or fragile substances • Immune recognition Ligand Regulatory site (one of four) Activator Binding site Active form Stabilized active form Oscillation Allosteric inhibitor Binding stabilizes inactive form protein Allosteric activators and inhibitors. In the cell, activators and inhibitors dissociate when at low concentrations. All binding proteins have similar characteristics: The enzyme can then oscillate again. 1. Specificity 2. Saturation Non-functional Inactive form Inhibitor Stabilized inactive form 3. Competitive Inhibition active site Figure 8.20 Heyer 4 Enzymes & Metabolism Coupled Reactions •Catalysis of one reaction allows the catalysis of a second reaction by a Product Coupled Reactions: Bioenergetics different active site on the same enzyme. B Product • Energy transfer from one molecule to another Substrate C couples chemical reactions Reaction 1 A • Exergonic reaction: reaction releases energy • Endergonic reaction: reaction requires energy Enzyme • Coupled bioenergetic reactions: the energy released by the exergonic reaction is used to power the Substrate endergonic reaction. Y Product Reaction 2 Z •Both reactions must occur for either to occur. Bioenergetics Free Energy & Entropy • More free energy (higher G) • Flow of energy in living systems obeys: • Less stable • At maximum stability • 1st law of thermodynamics: • Greater work capacity – The system is at equilibrium – Energy can be transformed, but it cannot be created or destroyed. A spontaneous change • The released free energy • 2nd law of thermodynamics: can be harnessed to do work – Energy transformations increase entropy (degree of . disorganization of a system). – Only free energy (energy in organized state) can be used • Less free energy (lower G) to do work. • More stable • Less work capacity (a) Gravitational motion. (b) Diffusion. Molecules (c) Chemical reaction. • Systems tend to go from states of higher free energy to states of Objects move spontaneously in a drop of dye diffuse In a cell, a sugar lower free energy. from a higher altitude to a until they are randomly molecule is broken lower one. dispersed. down into simpler Figure 8.5 molecules. Exergonic and Endergonic reactions in metabolism Coupled Reactions: Bioenergetics • An exergonic reaction • An endergonic reaction • Cells must maintain highly – Proceeds with a net release – Is one that absorbs free organized, low-entropy state at of free energy and is energy from its the expense of free energy. spontaneous surroundings and is – Cells cannot use heat for energy. nonspontaneous • Energy released in exergonic Reactants Products reactions used to drive endergonic reactions. Amount of energy Amount of – Require energy released in released energy exergonic reactions (ATP) to be (∆G <0) released Energy Energy (∆G>0) directly transferred to chemical- Products Reactants Free energy Free energy bond energy in the products of endergonic reactions. Progress of the reaction Progress of the reaction (a) Exergonic reaction: energy released (b) Endergonic reaction: energy required Figure 8.6 Heyer 5 Enzymes & Metabolism Endergonic and Exergonic The Role of ATP: Reactions Energy transfer from one molecule to • Endergonic: – Chemical reactions that another couples chemical reactions require an input of energy to make reaction “go.” • Products must contain A-P-P~P + glucose --> more free energy than reactants. glucose-6~phosphate + A-P-P • Exergonic: – Convert molecules with • Exergonic
Load more

Recommended publications
  • Amphibolic Nature of Krebs Cycle
    Amphibolic nature of Krebs Cycle How what we are is what we eat • In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes. • Since the citric acid does both synthesis (anabolic) and breakdown (catabolic) activities, it is called an amphibolic pathway • The citric acid cycle is amphibolic (i.e it is both anabolic and catabolic in its function). • It is said to be an AMPHIBOLIC pathway, because it functions in both degradative or catabolic and biosynthetic or anabolic reactions (amphi = both) A central metabolic pathway or amphibolic pathway is a set of reactions which permit the interconversion of several metabolites, and represents the end of the catabolism and the beginning of anabolism • The KREBS CYCLE or citric acid cycle is a series of reactions that degrades acetyl CoA to yield carbon dioxide, and energy, which is used to produce NADH, H+ and FADH. • The KREBS CYCLE connects the catabolic pathways that begin with the digestion and degradation of foods in stages 1 and 2 with the oxidation of substrates in stage 3 that generates most of the energy for ATP synthesis. • The citric acid cycle is the final common pathway in the oxidation of fuel molecules. In stage 3 of metabolism, citric acid is a final common catabolic intermediate in the form of acetylCoA. • This is why the citric acid cycle is called a central metabolic pathway. Anaplerosis and Cataplerosis Anaplerosis is a series of enzymatic reactions in which metabolic intermediates enter the citric acid cycle from the cytosol. Cataplerosis is the opposite, a process where intermediates leave the citric acid cycle and enter the cytosol.
    [Show full text]
  • Fructose Metabolism from a Functional
    SSE #174 Sports Science Exchange (2017) Vol. 28, No. 174, 1-5 FRUCTOSE METABOLISM FROM A FUNCTIONAL PERSPECTIVE: IMPLICATIONS FOR ATHLETES Luke Tappy, MD | Department of Physiology, Faculty of Biology and Medicine, University of Lausanne, Service of Endocrinology, Diabetes and Metabolism | Lausanne University Hospital, and Cardio-metabolic Center, Broye Hospital | Estavayer-le-lac, Switzerland • Fructose was originally a seasonal natural nutrient, mainly consumed in summer and fall in fruits and vegetables. In the industrial era, it became a permanent constituent of our diet, essentially a constituent of added sugars (sucrose, high-fructose corn syrup). • Fructose cannot be directly metabolized by most cells in our body. It has to be processed first in the gut, liver and kidneys, where it is converted into glucose, lactate and fatty acids. • Too much dietary fructose along with excess energy intake and low physical activity can cause hepatic insulin resistance, hypertriglyceridemia and increased hepatic fat content. GAT11LOGO_GSSI_vert_fc_grn • In exercising athletes, net carbohydrate oxidation increases with glucose ingestion in a dose-dependent manner until a plateau is reached at about 1g/min. The addition of fructose to glucose drinks can further increase carbohydrate oxidation. • During exercise, substantial amounts of fructose can be converted into lactate in splanchnic organs if available and released in the systemic circulation to be oxidized in contracting muscles. This “reverse fructose-lactate Cori cycle” provides additional energy substrate to muscle during exercise. • Conversion of fructose into glucose and lactate in splanchnic organs is associated with enhanced splanchnic energy expenditure, while muscle energy efficiency is minimally altered. • During recovery after exercise, glucose and fructose mutually enhance their gut absorption and their storage as glycogen in the liver.
    [Show full text]
  • • Glycolysis • Gluconeogenesis • Glycogen Synthesis
    Carbohydrate Metabolism! Wichit Suthammarak – Department of Biochemistry, Faculty of Medicine Siriraj Hospital – Aug 1st and 4th, 2014! • Glycolysis • Gluconeogenesis • Glycogen synthesis • Glycogenolysis • Pentose phosphate pathway • Metabolism of other hexoses Carbohydrate Digestion! Digestive enzymes! Polysaccharides/complex carbohydrates Salivary glands Amylase Pancreas Oligosaccharides/dextrins Dextrinase Membrane-bound Microvilli Brush border Maltose Sucrose Lactose Maltase Sucrase Lactase ‘Disaccharidase’ 2 glucose 1 glucose 1 glucose 1 fructose 1 galactose Lactose Intolerance! Cause & Pathophysiology! Normal lactose digestion Lactose intolerance Lactose Lactose Lactose Glucose Small Intestine Lactase lactase X Galactose Bacteria 1 glucose Large Fermentation 1 galactose Intestine gases, organic acid, Normal stools osmotically Lactase deficiency! active molecules • Primary lactase deficiency: อาการ! genetic defect, การสราง lactase ลด ลงเมออายมากขน, พบมากทสด! ปวดทอง, ถายเหลว, คลนไสอาเจยนภาย • Secondary lactase deficiency: หลงจากรบประทานอาหารทม lactose acquired/transient เชน small bowel เปนปรมาณมาก เชนนม! injury, gastroenteritis, inflammatory bowel disease! Absorption of Hexoses! Site: duodenum! Intestinal lumen Enterocytes Membrane Transporter! Blood SGLT1: sodium-glucose transporter Na+" Na+" •! Presents at the apical membrane ! of enterocytes! SGLT1 Glucose" Glucose" •! Co-transports Na+ and glucose/! Galactose" Galactose" galactose! GLUT2 Fructose" Fructose" GLUT5 GLUT5 •! Transports fructose from the ! intestinal lumen into enterocytes!
    [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]
  • Inhibition of Fructolytic Enzymes in Boar Spermatozoa by (S)-A-Chlorohydrin and L-Chloro-3-Hydroxypropanone
    Aust. J. BioI. Sci., 1986, 39, 395-406 Inhibition of Fructolytic Enzymes in Boar Spermatozoa by (S)-a-Chlorohydrin and l-Chloro-3-hydroxypropanone A. R. Jones, W. A. Bubb, S. R. Murdoch, and D. A. Stevenson Department of Biochemistry, The University of Sydney, New South Wales 2006. Abstract When boar spermatozoa were incubated with the (S)-isomer of the male antifertility agent a-chlorohydrin the activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited. The (R)-isomer had no significant effect on the activity of this enzyme whereas (R,S)-3-chlorolactaldehyde caused an inhibition of its activity and also in that of lactate dehydrogenase. The in vitro production of (S)-3-chlorolactaldehyde, the active metabolite of (S)-a-chlorohydrin, was attempted by incubating boar spermatozoa with l-chloro-3- hydroxypropanone. Preliminary results lead us to propose that this compound is converted into (S)-3- chlorolactaldehyde as well as to another metabolite which is an inhibitor of other enzymes within the fructolytic pathway. Introduction Of the many non-steroidal chemicals which are known to elicit an antifertility response in the male (Jackson 1966), only one compound has most of the attributes of an ideal male contraceptive. This compound, (S)-a-chlorohydrin [(S)-3-chloro­ propan-l,2-diol, I (Fig. 1)], affects the metabolic activity of mature spermatozoa by inhibiting the activity of the glycolytic or fructolytic enzyme glyceraldehyde- 3-phosphate dehydrogenase (Ee 1.2.1.12) thereby causing a decrease in the fructolytic flux. This limits the capability of the spermatozoa to synthesize ATP so that when CHpH I c=o I CH2C1 III Fig.
    [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]
  • Process for Purification of Ethylene Compound Having Fluorine-Containing Organic Group
    Office europeen des brevets (fi) Publication number : 0 506 374 A1 @ EUROPEAN PATENT APPLICATION @ Application number: 92302586.0 @ Int. CI.5: C07C 17/38, C07C 21/18 (g) Date of filing : 25.03.92 (30) Priority : 26.03.91 JP 86090/91 (72) Inventor : Kishita, Hirofumi 3-19-1, Isobe, Annaka-shi Gunma-ken (JP) (43) Date of publication of application : Inventor : Sato, Shinichi 30.09.92 Bulletin 92/40 3-5-5, Isobe, Annaka-shi Gunma-ken (JP) Inventor : Fujii, Hideki (84) Designated Contracting States : 3-12-37, Isobe, Annaka-shi DE FR GB Gunma-ken (JP) Inventor : Matsuda, Takashi 791 ~4 YdndSG Anri3k3~shi @ Applicant : SHIN-ETSU CHEMICAL CO., LTD. Gunma-ken (JP) 6-1, Ohtemachi 2-chome Chiyoda-ku Tokyo (JP) @) Representative : Votier, Sidney David et al CARPMAELS & RANSFORD 43, Bloomsbury Square London WC1A 2RA (GB) (54) Process for purification of ethylene compound having fluorine-containing organic group. (57) A process for purifying an ethylene compound having a fluorine-containing organic group (fluorine- containing ethylene compound) by mixing the fluorine-containing ethylene compound with an alkali metal or alkaline earth metal reducing agent, and subjecting the resulting mixture to irradiation with ultraviolet radiation, followed by washing with water. The purification process ensures effective removal of iodides which are sources of molecular iodine, from the fluorine-containing ethylene compound. < h- CO CO o 10 o Q_ LU Jouve, 18, rue Saint-Denis, 75001 PARIS EP 0 506 374 A1 BACKGROUND OF THE INVENTION 1. Field of the Invention 5 The present invention relates to a process for purifying an ethylene compound having a fluorine-containing organic group, and more particularly to a purification process by which iodine contained as impurity in an ethylene compound having a fluorine-containing organic group can be removed effectively.
    [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]
  • Metabolism and Energetics Oxidation of Carbon Atoms of Glucose Is the Major Source of Energy in Aerobic Metabolism
    Metabolism and Energetics Oxidation of carbon atoms of glucose is the major source of energy in aerobic metabolism C6H1206 + 6O2 yields 6 CO2 + H20 + energy Energy released ΔG = - 686 kcal/mol Glucose oxidation requires over 25 discrete steps, with production of 36 ATP. Energy Transformations The mitochondrial synthesis of ATP is not stochiometric. Electron–motive force Proton-motive force Phosphoryl-transfer potential in the form of ATP. Substrate level phosphorylation The formation of ATP by substrate-level phosphorylation ADP ATP P CH O P CH2 O P 2 is used to represent HC OH HC OH a phosphate ester: phosphoglycerate CH OH OH CH2 O P kinase 2 P O bisphosphoglycerate 3-phosphoglycerate OH ADP ATP CH2 CH2 CH3 C O P C OH C O pyruvate kinase non-enzymic COOH COOH COOH phosphoenolpyruvate enolpyruvate pyruvate Why ATP? The reaction of ATP hydrolysis is very favorable ΔGo = - 30.5 kJ/mol = - 7.3 kCal/mol 1. Charge separation of closely packed phosphate groups provides electrostatic relief. Mg2+ 2. Inorganic Pi, the product of the reaction, is immediately resonance-stabilized (electron density spreads equally to all oxygens). 3. ADP immediately ionizes giving H+ into a low [H+] environment (pH~7). 4. Both Pi and ADP are more favorably solvated by water than one ATP molecule. 5. ATP is water soluble. The total body content of ATP and ADP is under 350 mmol – about 10 g, BUT … the amount of ATP synthesized and used each day is about 150 mol – about 110 kg. ATP Production - stage 1 - Glycolysis Glycolysis When rapid production of ATP is needed.
    [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]