* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Vitamins and Coenzymes - KSU - Home
Deoxyribozyme wikipedia , lookup
Metabolic network modelling wikipedia , lookup
Photosynthesis wikipedia , lookup
Electron transport chain wikipedia , lookup
Microbial metabolism wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Catalytic triad wikipedia , lookup
Radical (chemistry) wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biosynthesis wikipedia , lookup
Lactate dehydrogenase wikipedia , lookup
Citric acid cycle wikipedia , lookup
Biochemistry wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Nicotinamide adenine dinucleotide wikipedia , lookup
King Saud University College of Science Department of Biochemistry Disclaimer • The texts, tables and images contained in this course presentation are not my own, they can be found on: – References supplied – Atlases or – The web Part 1 Coenzyme-Dependent Enzyme Mechanism Professor A. S. Alhomida 1 Syllabus • Instructor: Professor A. S. Alhomida – Office: 2A 62; Tel: 467-5938 – E-mail: [email protected] – Web page: faculty.ksu.edu.sa/alhomida • Textbook: 1. Enzyme kinetics and mechanism. Cook and Cleland, 2007 2. Enzymatic Reaction Mechanisms. Walsh, 1979 3. Introduction to Enzyme and Coenzyme Chemistry, 2nd Edition. Bugg, 2004 4. Contemporary Enzyme Kinetics and Mechanism. Purich, 1983 5. Structure and Mechanism in Protein Science: A guide to Enzyme Catalysis and Protein folding. Fersht, 1999 6. Biochemistry 2nd edition. Garrett and Grisham, Chapter, 14-16 2 Syllabus, Cont’d Lechninger's Principles of Biochemistry 4th edition. D. L. Nelson and M.M. Cox., Chapter 6 8. Biochemistry 3rd Edition. Mathews, Holde and Ahern. Chapter: 11 9. Fundamentals of Biochemistry 2nd Edition by Voet and Voet. Chapters: 11, 12 10. Biochemistry 3rd Edition. Zubay. Chapters: 8-11 11. Stryer’s Biochemistry, 5th Edition. Berg, Tymoczko and Stryer. Chaper 8-10. 12. Principles of Biochemistry, 4th Edition. Horton, Scrmgeour, Perry and Rawn. Chapter 5-7 7. 3 Oral Presentation Project • It will focus on article dealing with enzyme mechanisms and you will give a short oral presentation to the class on your analysis of the article • This project is designed to give you some experience with reading and interpreting original research reports that deal with the study of enzymatic reaction mechanisms and with making an oral presentation of a scientific study using PowerPoint 4 Oral Presentation Project, Cont’d • Choose an article from a current issue of a biochemical journal • The article must be an original research report (not a review article) that deals with the study of the mechanism of action of some enzyme and utilizes the techniques of sitedirected mutagenesis or site-directed inactivation (transition state analogs) 5 Oral Presentation Project, Cont’d • Prepare and give a 10-15 minutes oral presentation that gives an overview of the study described in your paper and an explanation of the data that supports the author’s conclusions • The presentations will be given in class on December 22nd • Submit a soft copy of your presentation saved in CD disk using PowerPoint 6 Oral Presentation Project, Cont’d • Completion Schedule – On Saturday, December 22nd, select a date for your presentation – Before Saturday, January 5th, submit a photo copy of your chosen article. I will make copies of articles and distribute them to other members of the class on Saturday, January 12th • The project will account for 20 out of 70 of your course grade 7 8 Vitamins and Coenzymes 9 10 11 12 13 14 Vitamins in Metabolic Pathways Glycogenolysis Glc PP a vit B6 G1P Glycogen Ala Asp Glycolysis PPP G6P ALT vit B6 R5P TK vit B1 G3P Pyr PDH vit B1,B2,B3 Acetyl-CoA AST vit B6 OA TCA cycle SCoA aKG vit B6 aKGDH vit B1,B2,B3 Glu 15 Coenzymes and Vitamins • Some enzymes require cofactors for activity (1) Essential ions (mostly metal ions) (2) Coenzymes (organic compounds) Apoenzyme + Cofactor (protein only) Holoenzyme (active) (inactive) 16 Coenzymes • Coenzymes act as group-transfer reagents • Hydrogen, electrons, or other groups can be transferred • Larger mobile metabolic groups can be attached at the reactive center of the coenzyme • Coenzyme reactions can be organized by their types of substrates and mechanisms 17 Types of cofactors 18 Inorganic Cations • Enzymes requiring metal ions for full activity: (1) Metal-activated enzymes have an absolute requirement or are stimulated by metal ions (examples: K+, Ca2+, Mg2+) (2) Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt ) 19 Carbonic Anhydrase 20 Carbonic Anhydrase • Carbon dioxide (CO2) is a major end product of aerobic metabolism • In mammals, this CO2 is released into the blood and transported to the lungs for exhalation • While in the blood, CO2 reacts with water • The product of this reaction is a moderately strong acid, carbonic anhydride (pKa = 3.5), which becomes bicarbonate ion on the loss of a H+ 21 Carbonic Anhydrase, Cont’d • Almost all organisms contain enzyme, carbonic ahydrase, that catalyzes the below reaction • Cabonic anhydrase accelerates CO2 hydration dramatically at rate as high as kcat = 106 s-1 22 Types of Carbonic Anhydrases • a-Carbonic anydrase: – Found in human, animals, some bacteria and algae – Trimer • b-Carbonic anydrase – Higher plants and many bacteria and E. coli – Has only one conserved His whereas in a has three His • g-Carbonic anhydrase – Found in bacteria Methanoscarcina thermophila – Has three zn sites similar to a-carbonic anhydrase 23 Structure of a-Carbonic Anydrase • Zn2+ is coordinated by the imidazole rings of three His residues, His-94, His-96 and His119 • The primary function of the enzyme in animals is to interconvert CO2 and bicarbonate to maintain acid-base balance in blood and other tissues and to help transport CO2 out of tissues 24 Structure of β-Carbonic Anhydrase • Found in plans which is an evolutionarily distinct enzyme but participates in the same reaction and also uses a Zn2+ in its active site • It helps raise the concentration of CO2 within the chloroplast to increase the carboxylation rate of the enzyme Rubisco • It integrates CO2 into organic carbon sugars during photosynthesis, and can only use the CO2 form of carbon, not carbonic acid nor bicarbonate 25 Structure of a-Carbonic Anydrase Three subunits Zn bound to three His 26 Structure of g-Carbonic Anhydrase • (Left) the Zn site, (Middle) the trimeric structure (A, B, and C) and (Right) the enzyme is rotated to show a top-down view position of the Zn sites 27 Human carbonic anhydrase 28 Carbonic Anhydrase, Cont’d • How does this Zn2+ complex facilitates CO2 hydration? • A major clue comes from the pH profile of the enzymatic ally catalyzed CO2 hydration: 29 Carbonic Anhydrase, Cont’d • At pH 8, the reaction proceeds near its maximal rate • As the pH decreases, the rate of the reaction drops • The midpoint of this transition is near pH 7, suggesting that a group with pKa = 7 plays an important role in the activity of this enzyme 30 Carbonic Anhydrase, Cont’d • The deprotonated (high pH) form of this group participates more effectively in the catalysis • Although His have pKa value near 7, a variety of evidence suggest that the group responsible for this transition is not His but it is the Zn2+-bound water molecule • The binding of water to the positively charged Zn2+ center reduces the pKa of the water from 15.7 to 7 31 Carbonic Anhydrase, Cont’d 32 Carbonic Anhydrase, Cont’d • The lowered pKa generates Zn2+-OHcomplex that is sufficiently nucleophilic to attack CO2 more readily than water does 33 Mechanism of Carbonic Anhydrase 34 Mechanism of Carbonic Anhydrase His His His His His Zn2+ His Zn2+ B: O B: O H H O C O CO2 35 Mechanism of Carbonic Anhydrase, Cont’d • Zn2+ ion promotes the ionization of bound H2O. Resulting nucleophilic OH- attacks carbon of CO2 • The pKa of water drops from 15.7-7 when it is coordinate to Zn2+ • HO- is 4 orders of magnitude more nucleophlic than is water 36 His His His His His His Zn2+ 2+ Zn B: O O H H O H C H2O C O B: O O H O 37 His His His His His Zn2+ Zn2+ BH O H O O O C BH H H O Tetrahedral intermediate His O HO C O Bicarbonate 38 Mechanism of Carbonic Anhydrase, Cont’d 1. The important of Zn2+-OH- comlpex suggests a simple mechanism of CO2 hydration: 2. Zn2+ facilitates the release of a H+ from water, which generates a OH3. The CO2 binds to the enzyme’s active site and is positioned to react with the OH- 39 Mechanism of Carbonic Anhydrase, Cont’d 3. The OH- attacks nucleophilically CO2, converting it into bicarbonate ion 4. The catalytic site is regenerated with release of the bicarbonate ion and the binding of another molecule of water 40 Iron in Metalloenzymes • Iron undergoes reversible oxidation and reduction: Fe3+ + e- (reduced substrate) Fe2+ + (oxidized substrate) • Enzyme heme groups and cytochromes contain iron 41 Iron in Metalloenzymes, Cont’d • Nonheme iron exists in iron-sulfur clusters (iron is bound by sulfide ions and S- groups from cysteines) • Iron-sulfur clusters can accept only one e- in a reaction 42 Iron-sulfur clusters • Iron atoms are complexed with an equal number of sulfide ions (S2-) and with thiolate groups of Cys side chains 43 Coenzyme Classification • There are two classes of coenzymes (1) Cosubstrates are altered during the reaction and regenerated by another enzyme (2) Prosthetic groups remain bound to the enzyme during the reaction, and may be covalently or tightly bound to enzyme 44 Classification of Coenzymes in Mammals (1) Metabolite coenzymes- synthesized from common metabolites (2) Vitamin-derived coenzymes- derivatives of vitamins (vitamins cannot be synthesized by mammals, but must be obtained as nutrients) 45 Metabolite Coenzymes 46 Metabolite Coenzymes • Nucleoside triphosphates are examples 5`-C ATP g b a 47 Reactions of ATP • ATP is a versatile reactant that can donate its: (1) Phosphoryl group (g-phosphate) (2) Pyrophosphoryl group (g,b phosphates) (3) Adenylyl group (AMP) (4) Adenosyl group 48 • Nucleotide-sugar coenzymes are involved in carbohydrate metabolism • UDP-Glucose is a sugar coenzyme. It is formed from UTP and glucose 1-phosphate (UDP-glucose product next slide) 49 50 Carbon-Carbon Bond Formation 51 Alkylation Reactions • Methylation is an important transformation in the biosynthesis of many secondary metabolites • Organic chemists use methyl iodide or methyl sulphonates for methylations • The biological equivalent is S-adenosyl methionine (SAM) • The driving force for methyl group transfer is the conversion of a sulphonium ion into a neutral sulphide 52 Alkylation Reactions, Cont’d 53 Aldol and Claisen Reactions • Reactions between enolates (and their equivalents) with aldehydes or ketones are referred to as aldol reactions • Reaction of enolates with esters are referred to as Claisen reactions • They are the most common method to form carbon-carbon bonds • The biological equivalent of enolates are enamines and coenzyme A • These are co-factors of aldolase enzymes 54 Enamines • The side chain of the amino acid lysine carries an amino group • Reaction with carbonyl compounds leads to imines which tautomerise to give enamines • Enamines are enolate equivalents and react with carbonyl compounds through nucleophilic attack via their b-carbon • They are used in a very similar way in organic chemistry as shown below for the reaction of a secondary amine (pyrrolidin) with a ketone 55 Enamines, Cont’d 56 Aldol Reactions • Aldol Reactions Require Several Levels of Control: • Enol versus carbonyl component: • carbonyl compounds with acidic a-protons can either be deprotonated and react as nucleophiles, or react as electrophiles through their carbonyl group • If this is not carefully controlled an intractable mixture of products (cross-aldol products) is obtained 57 Aldol Reactions, Cont’d • Formation of an enamine avoids this problem • The enamine is only nucleophilic • Regioselectivity: enamines are ambident nucleophiles • They can in principle react through the carbon or the nitrogen atom • For aldol-type processes, only reactions through the carbon atom lead to the desired product 58 Aldol Reactions, Cont’d • In biological systems the regioselectivity is controled by the steric environment of the enzyme active site 59 Aldol Reactions, Cont’d • Stereoselectivity: The stereochemistry of aldol reactions is highly complex-syn, anti, matched case, mismatched case-are just a few keywords highlighting how difficult it is to control the relative and absolute stereochemistry of aldol products • In biological systems this is again taken care of by the stereochemistry of the active site of an enzyme 60 S-Adenosylmethionine (SAM) 61 SAM Biosynthesis • ATP is a source of other metabolite coenzymes such as S-adenosylmethionine (SAM) • SAM donates methyl groups in many biosynthesis reactions Methionine + ATP SAM + Pi + PPi 62 Structure of SAM • Activated methyl group in red 63 Functions of SAM 1. SAM donates the methyl group for many methylation reactions: Methylation of norepinephrins 64 Functions of SAM, Cont’d 2. SAM involves in redox radical-dependent enzymes: Pyruvate formate lyase; Anerobic ribonucleotide reductase 3. Until this point, the only know role for SAM was for methyl group transfer, thus it was surprising to find SAM involved in redox biochemistry 65 Functions of SAM, Cont’d 4. The involvement of SAM in redical biochemistry was first established for Lys 2,3-aminomustase (from C. subterminale) which converts Lys with b-Lys 5. Lys 2,3-aminomustase catalyzes the reaction by 1,2 rearrangement mechanism similar to Vit B12-dependent mutase, but didn’t use Vit B12 instead required PLP and SAM for activity and a reduced [4Fe4S] cluster 66 SAM as Methyl Group Donor – Methylation of bases in tRNA – Methylation of cytosine residues in DNA – Methylation of norepinephrine 67 SAM Cycle 1. SAM synthase (Met adenosyl transferase) 2. Methyltransferase 3. S-adenosyl homocysteinase 4. Homocysteine methyltransferase 68 Mechanism of SAM Synthase (Met Adenosyl Transferase) 69 Mechanism of SAM Synthase Unusual displacement of triphosphate reaction NH2 H N N H3N C CH2 COO O O 2 O P O P O P O O N N O CH2 O H S: CH3 O O H H Nucleophilic attack (SN2 mechanism) H OH OH ATP Methionine 70 Mechanism of SAM Synthase, Cont’d • Met is not a sufficient reactive to be a good methyl donor because of the homosysteine mercaptide anion is a poor leaving group • SAM synthase catalyzes an unusual displacement reaction because of Met sulfur atom attacks nucleophilically on the 5` carbon of ATP to produced the sulfonium compound and and inorganic triphosphate (PPPi ) is formed 71 Mechanism of SAM Synthase Supernucleophile Very good leaving group because of positively charged of S atom H H3N NH2 COO C N N SAM synthase CH2 2 N N S CH2 H CH3 O H H H OH OH SAM O O O O P O P O P O O PPPi O O O O P O P O H2 O O O PPi O O O O 2 O P O O Pi P O O 72 Mechanism of SAM Synthase, Cont’d • PPPi is then hydrolyzed by the same enzyme into PPi and Pi making the reaction thermodynamically more favorable • This is one of two reactions in which a displacement of this kind is known to occur in biological system 73 Mechanism of SAM Synthase, Cont’d • The other being the formation of adenosylcobalamin • The hydrolysis of PPPi drives the reaction to right highly exergoic in the synthetic direction 74 SAM-Dependent Methyltransferase 75 SAM-Dependent Methyltransferase • The functional roles of methylation are wide ranging and include biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repairing, nucleic acid processing, gene silencing and imprinting • The majority of methylation reactions are carried out by the SAM-dependent methyltransferases 76 SAM-Dependent Methyltransferase, Cont’d • Human thiopurine Smethyltransferase (TPMT) in complex with SAH • TPMT is a cytosolic drug-metabolizing enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, azathioprine, 6thioguanine 77 SAM-Dependent Methyltransferase, Cont’d • All methylation reactions requiring SAM are simple SN2 (Substitution of nucleophilic bimolecular) displacements • SAH is a potent inhibitor of all reactions in which a methyl group is transferred from SAM to an acceptor • It is important to prevent the accummulation of SAH in cells 78 SAM-Dependent Methyltransferase, Cont’d • This is accomplished through the action of Sadenosylhomocysteinase that converts SAH into adenosine and homocysteine • Homocysteine is converted into Met and adenosine (Ado) into inosine (via SAM cycle) 79 Mechanism of SAM-Dependent Methyltransferase 80 Mechanism of SAM-Dependent Methyltransferase H H3N HO CH2CH2N ..H2 HO OH NH2 COO C N CH2 2 S N N CH2 Norepinephrine Nucleophilic attack (SN2 Mechanism) N CH3 SAM O H H H H OH OH 81 Mechanism of SAM-Dependent Methyltransferase, Cont’d H H3N CH3 HO CH2CH2NH2 HO OH Epinephrine C NH2 COO N N + CH2 S 2 N N CH2 H O H H H OH OH S-adenosylhomocysteine (SAH) 82 SAM-Dependent Radical Enzymes 83 SAM-Dependent Radical Enzymes • Organic radicals are used by a number of enzymes to catalyze biochemical transformations with high-energy barriers that would be difficult to accomplish through nonradical heterolytic chemistry • Well known examples include: – Reduction of an alcohol to an alkane catalyzed by ribonucleotide reductase – Carbon chain rearrangements catalyzed by methylmalonyl CoA mutase or glutamate mutase 84 SAM-Dependent Radical Enzymes • Organic radicals can be generated in enzymes through only three general mechanisms: – Metal-activated oxygen biochemistry – Adenosylcobalamin (Vit B12) biochemistry, or – Reduction of the sulfonium of SAM 85 Pyruvate Formate Lyase (Formate C-Acetyltransferase) 86 Pyruvate Formate Lyase • It is an important enzyme (found in Escherichia coli and other organisms) that helps regulate anaerobic glucose metabolism • Using radical biochemistry, it catalyzes the reversible conversion of pyruvate and CoA into formate and acetyl-CoA 87 Structure of Pyruvate Formate Lyase • It is a homodimer made of 85 kD, 759-residue subunits • It has a 10-stranded b/a barrel motif into which is inserted a b finger that contains major catalytic residues • The active site of the enzyme, elucidated by X-ray crystallography, holds three essential amino acids that perform catalysis: – Gly-734 – Cys-418 – Cys-419 88 Structure of Pyruvate Formate Lyase, Cont’d • It is a homodimeric protein (2 x 85 kD) and catalytically inactive when isolated • Activated enzyme contains one protein radical per dimer at Gly734 and has a half of the sites reactivity 89 Structure of Pyruvate Formate Lyase, Cont’d • Three major residues that hold the substrate pyruvate close by Arg-435, Arg-176, and Ala272), and two flanking hydrophobic residuesTrp-333 and Phe-432 • The active site of enzyme is a similar to that of class I and class III ribonucleotide reductase 90 SAM-[4Fe4S] Cluster SAM • The interaction of SAM with the [4Fe–4S]1+ of activated en\yme • a-N and a-carboxyl O of Met anchors the SAM to the cluster with the sulfonium interacting with a sulfide from the cluster a [4Fe4S] cluster 91 Regulationn of Pyruvate Formate Lyase Radical Radical Gly-734 (AE) Activase (DE) Deactivase 92 Reaction of Pyruvate Formate Lyase H N O 734 H N H Gly-734 Pyruvate formate lyase H H N O 734 N H [4Fe4S] red + SAM Gly-734 radical 93 Reaction of Pyruvate Formate Lyase, Cont’d Pyruvate formate lyase H O N 734 O Gly-734 radical N H O O H H3C O CoA O Formate O Pyruvate H3C S Acetyl-CoA CoA 94 Mechanism of Pyruvate Formate Lyase 95 Role of Catalytic Residues Gly-734 (glycyl radical) – Transfers the radical on and off Cys-418, via Cys419 • Cys-418 (thiyl radical) – Does acylation reaction on the carbon atom of the pyruvate carbonyl • Cys-419 (thiyl radical) – Performs hydrogen-atom transfers 96 Generation of 5`-deoxyadenosyl Radical from SAM by [4Fe4S] Cluster 5`-deoxyadenosyl radical 2 Ad Ad O O Enz OH H3C S Fe S S S H S H3C S OH Fe S O O SAM OH H2C Fe Fe Fe H2 N Enz S S H OH Fe O Fe Fe H2 N O S 97 Mechanism of Pyruvate Formate Lyase 98 Mechanism of Pyruvate Formate Lyase Gly-734 Cys-419 H Radical transfer from Gly-734 to Cys-419 Cys-418 S H S 99 Mechanism of Pyruvate Formate Lyase, Cont’d Gly-734 Gly-734 Cys-419 H H S H Cys-419 Cys-418 H H H S Cys-418 S O O Pyruvate Radical transfer from Cys-419 to Cys-418 S H3C O 100 Mechanism of Pyruvate Formate Lyase, Cont’d Gly-734 Gly-734 Cys-419 Cys-419 H H H S H H H Cys-418 Cys-418 O S O S S H3C O O Tetrahedral radical intermediate O H3C formate radical intermediate O Thioester (acyl-enzyme) 101 Mechanism of Pyruvate Formate Lyase, Cont’d Gly-734 Cys-419 Radical transfer from Cys-419 to CoA S H H Cys-418 S CoA-S H H3C CoA-S H O O H O Formate 102 Mechanism of Pyruvate Formate Lyase, Cont’d Gly-734 Gly-734 Cys-419 H H H Cys-419 S Cys-418 H H H S Cys-418 S S CoA-S CoA-S Radical transfer from CoA to acetate H3C O H3C O Tetrahedral radical intermediate 103 Mechanism of Pyruvate Formate Lyase, Cont’d Gly-734 Cys-419 H H H S Cys-418 S O CoA-S Radical Cys-418 CH3 Acetyl-CoA 104 Mechanism of Pyruvate Formate Lyase, Cont’d Gly-734 Gly-734 Cys-419 H H H Cys-419 S H Cys-418 S Cys-418 radical enzyme e H H S Cys-418 H S Cys-418 radical inactivated enzyme 105 Mechanism of Pyruvate Formate Lyase, Cont’d 1. The proposed mechanism begins with radical transfer from Gly-734 to Cys-418, via Cys-419 2. The Cys-418 thiyl radical adds covalently to C-2 of pyruvate, generating an acetylenzyme intermediate (which now contains the radical) 3. The acetyl-enzyme intermediate releases a formyl radical that undergoes hydrogenatom transfer with Cys-419 106 Mechanism of Pyruvate Formate Lyase, Cont’d 4. CoA comes in and undergoes hydrogenatom transfer with the Cys-419 radical to generate a CoA radical 5. The CoA radical then picks up the acetyl group from Cys-418 to generate acetyl-CoA, leaving behind a Cys-418 radical 6. Enzyme can then undergo radical transfer to put the radical back onto Gly-734 7. Note that each step is reversible 107 Mechanism for Generating Radical Gly-734 From favorodoxin Trasfer radical to inactivated Gly-724 enzyme 108 Mechanism for Generating Radical Gly734 1. Activated enzyme has a novel radical mechanism that utilizes an Fe–S cluster and SAM to facilitate generation of a putative adenosyl radical 2. The Fe–S cluster has a unique iron site in the [4Fe–4S] cluster which is used to coordinate an amino a-nitrogen and acarboxyl oxygen to anchor SAM in the active site 109 Mechanism for Generating Radical Gly734, Cont’d 3. Inner-sphere electron transfer from a bridging sulfide of the [4Fe–4S]1+ cluster to the sulfonium of SAM (AdoMet) causes C–S bond homolysis, which produces a 5′deoxyadenosyl radical and Met 4. This anchoring allows for the potential innersphere electron transfer from the bridging sulfide to the sulfonium of SAM, and facilitates homolytic bond cleavage and creation of the adenosyl radical 110 Mechanism for Generating Radical Gly734, Cont’d 5. The adenosyl radical abstracts a hydrogen from Gly-734 of enzyme and 5′deoxyadenosine and Met are replaced with another SAM 6. The active cluster of enzyme has to be in reduced form ([4Fe–4S]1+), which is oxidized to [4Fe–4S]2+ during turnover catalysis 7. The source of the electron is proposed to be a reduced flavodoxin 111 Vitamin-Derived Coenzymes 112 Vitamin-Derived Coenzymes • Vitamins are required for coenzyme synthesis and must be obtained from nutrients • Animals rely on plants and microorganisms for vitamin sources (meat supplies vitamins also) • Most vitamins must be enzymatically transformed to the coenzyme 113 Vitamin C 114 Vitamin C: a Vitamin but not a Coenzyme • A reducing reagent for hydroxylation of collagen • Deficiency leads to the disease scurvy • Most animals (not primates) can synthesize Vit C 115 Vitamin C (ascorbic acid) in Foods 116 Nicotinamide Adenine Dinucleotide 117 Niacin in Foods 118 Niacin in Foods 119 Reduction Reactions • The biological equivalent of hydride transfer reagents, such as NaBH4, is nicotinamide adenine dinucleotide (NADH) and its phosphorylated analog NADPH • These are coenzymes of reductase enzymes • The stick model of NAD is taken from an actual X-ray crystallographic analysis of human alcohol dehydrogenase enzyme 120 Reduction Reactions, Cont’d 121 Reduction Reactions, Cont’d • The pyridinium ring acts as hydride acceptor in the oxidation step, whilst 1,4dihydropyridine system acts as hydride donor in the reduction step: 122 Reduction Reactions, Cont’d • The stereoselectivity of the reduction step relies on the "chiral environment" provided by the active side of the enzyme • NADH is a coenzyme which is held in the acitve site of the enzyme (alcohol dehydrogenase in this case) by non-covalent interactions • The image below shows NADH and amino acids in a distance of 5 Å from NADH 123 Reduction Reactions, Cont’d 124 Reduction Reactions, Cont’d • The image on the left is a close-up view of the residues neighbouring NADH in the active site • The image on the right shows the whole enzyme (the enzyme is actually a dimer and only one half is shown for clarity) 125 Oxidation Reactions • NAD-dependant Enzymes • Oxidation is the reverse of reduction and the oxidized form of NADH can act as an oxidant • In oxidation-mode NAD/NADH-dependant enzymes are referred to as oxidase enzymes • This form is called NAD 126 Oxidation Reactions, Cont’d • In fact, NAD and NADH have to be reversible redox pairs to allow the coenzyme and the enzyme to act as true catalysts 127 Cytochrome-P450-dependant Enzymes • The redox-active species in this class of enzymes is the Fe(III)-Fe(II) couple • The iron centre is coordinated to a porphorine system • Together they form the hem coenzyme of oxygenase enzymes (note the difference to oxidase enzymes which contain NAD as coenzyme) • The name cytochrome P450 is due to the strong absorption at 450 nm of enzymes that contain a hem coenzyme when co-ordinated to carbon monoxide 128 Cytochrome-P450-dependant Enzymes, Cont’d 129 Non-Hem a-Ketoglutarate-Dependant Oxygenases • Enzymes belonging to this class contain an iron centre, but no hem coenzyme • Isopenicillin-N-synthase, the crucial enzyme in the biosynthesis of penicillin belongs to this class 130 NAD+ and NADP+ • Nicotinic acid (niacin) is precursor of NAD and NADP • Lack of niacin causes the disease pellagra • Humans obtain niacin from cereals, meat, legumes 131 Oxidized, reduced forms of NAD (NADP) 132 Structure of NAD 133 NAD and NADP are cosubstrates for dehydrogenases • Oxidation by pyridine nucleotides always occurs two electrons at a time • Dehydrogenases transfer a hydride ion (H:-) from a substrate to pyridine ring C-4 of NAD+ or NADP+ • The net reaction is: NAD(P)+ + 2e- + 2H+ NAD(P)H + H+ 134 Biosynthesis of NAD(P) 135 Oxidoreductase and Dehydrogenase 136 Oxidoreductase and Dehydrogenase • Oxidoreductases that transfer electron from one molecule to another • These enzymes catalyze the oxidation reaction: A(red) + B(oxid) A(oxid) + B(red) • In reality, free electrons do not exists as these reactions involve atoms transfer 137 Oxidoreductase and Dehydrogenase • Dehydrogenases: that involve removing hydrogen from the electron donor during metabolic oxidation reactions • Oxidases are used only for the enzymes in which the oxidation reaction with molecular oxygen (O2) participating as the electron acceptor 138 Dehydrogenase Nomenclature • The common scheme for making names for oxidoreductases is adding donor name to the dehydrogenase, i.e. donor dehydrogenase. • For example: alcohol dehydrogenase, lactate dehydrogenase, etc • The proper name consists from the donor name, acceptor name together with oxidoreductase, i.e. donor: acceptor oxidoreductase 139 Dehydrogenase Nomenclature • Sometimes the construction acceptor reductase is used: – Example: Enzyme EC 1.1.1.1 Systematic name: alcohol:NAD+ oxidoreductase Accepted name: alcohol dehydrogenase 140 Enzymatic Classification of Dehydrogenases • According to the Enzyme Nomenclature from NC-IUBMB the nomenclature and classification of enzymes is based on the reaction they catalyze • Each reaction, catalyzed by enzyme is specified by the Enzyme Commission number or EC number 141 Enzymatic Classification of Dehydrogenases • Each EC number consists of the EC and for digits separated by periods • Each digit represents the progressively higher level of enzyme classification • Dehydrogenases are belongs to the EC 1 Oxidoreductases group • Oxidoreductases classification according to the substrate they utilize: 142 • • • • • • • • • • • • EC 1.1 - Acting on the CH-OH group of donors EC 1.2 - Acting on the aldehyde or oxo group of donors EC 1.3 - Acting on the CH-CH group of donors EC 1.4 - Acting on the CH-NH2 group of donors EC 1.5 - Acting on the CH-NH group of donors EC 1.6 - Acting on NADH or NADPH EC 1.7 - Acting on other nitrogenous compounds as donors EC 1.8 - Acting on a sulfur group of donors EC 1.9 - Acting on a heme group of donors EC 1.10 - Acting on diphenols and related substances as donors EC 1.11 - Acting on a peroxide as acceptor EC 1.12 - Acting on hydrogen as donor 143 • EC 1.13 - Acting on single donors with incorporation of molecular oxygen (oxygenases) • EC 1.14 - Acting on paired donors, with incorporation or reduction of molecular oxygen • EC 1.15 - Acting on superoxide as acceptor • EC 1.16 - Oxidizing metal ions • EC 1.17 - Acting on CH or CH2 groups • EC 1.18 - Acting on iron-sulfur proteins as donors • EC 1.19 - Acting on reduced flavodoxin as donor • EC 1.20 - Acting on phosphorus or arsenic in donors • EC 1.21 - Acting on X-H and Y-H to form an X-Y bond • EC 1.97 - Other oxidoreductases • EC 1.98 - Enzymes using H2 as reductant • EC 1.99 - Other enzymes using O2 as oxidant 144 Structural Classification of Dehydrogenases • Currently, two different classifications of dehydrogenases are exists: – One is historical for polyol dehydrogenases and – Another is modern UniProt protein classification for dehydrogenases and oxydoreductases • You still can use ancient classification, but it is necessary to remember, that these classification are slightly different • Please also remember, that alcohol dehydrogenase classification is slightly inconsistent 145 Dehydrogenase Catalytic Mechanism • Dehydrogenases transfer protons to an acceptor or coenzymes such as NAD+/NADH or NADP+/NADPH, FAD/FMN • The wide diversity of dehydrogenases does not allow to develop a uniform catalytic mechanism for all cases • All NAD+/NADH reactions in the body involve 2 electron hydride transfers 146 Dehydrogenase Catalytic Mechanism • NAD+/NADH can undergo two electron redox steps, in which a hydride is transferred from a substrate to the NAD+, with the electrons flowing to the positively charged nitrogen of NAD+ which serves as an electron sink 147 148 Dehydrogenase Catalytic Mechanism • NADH does not react well with dioxgyen (O2) • Since single electron transfers to/from NAD+/NADH produce free radical species which can not be stabilized effectively 149 Dehydrogenase Catalytic Mechanism 150 Hydrogenases • The enzymes that catalyze hydrogen production are hydrogenases (not dehydrogenses) • Crystal structures of hydrogenases show them to be unique among metal-containing enzymes • They contain two metals bonded to each other. The metal centers can either be both iron or one each of iron and nickel 151 Experimental Evidences for Hydride Ion Transfer 152 Alcohol Dehydrogenase H H CONH2 CONH2 CH3CH2OH + N N O H3C C H R R NADH NAD • if run in T2O or D2O, no T or D incorporation in NADH • if run with H3CCD2OH, complete D incorporation in NADH • Results consistent with a hydride-transfer (H-) mechanism and not a proton-transfer (H+) Enzyme Enzyme H B H3C :B H C O H H H3C C O H H H CONH2 CONH2 CH3CH2OH N N R R NAD NADH 153 H H CONH2 ADH CH3CH2OH N R NAD CONH2 Ethanol N R O + H3C C H Acetaldehyde NADH 1. If run in T2O or D2O, no T or D incorporation in NADH 2. If run with H3CCD2OH, complete D incorporation in NADH 154 3. Results consistent with a hydride-transfer (H-) mechanism and not a proton-transfer (H+) Enzyme Enzyme H B H3C :B H C O H H H3C C O H H H CONH2 CONH2 CH3CH2OH N N R R NAD NADH 155 Experimental Evidence for a Hydride-transfer vs an Electrontransfer mechanism • Cyclopropyl carbinyl radical ring opening as a probe for radical intermediates k ~ 108 s-1 cyclopropyl carbinyl radical (radical clock) 4-butenyl radical 156 Experimental Evidence for a Hydride-transfer vs an Electron-transfer mechanism 157 lactate dehydrogenase O CO2H NADH OH CO2H 2˚ alcohol lactate dehydrogenase O OH CO2H CO2H NADH 2˚ alcohol Product consistent with a hydride-transfer mechanism 158 • If an electron-transfer mechanism: + e- O O CO2H CO2H O 2 H+ CO2H + e- O CO2H O CO2H a- keto acid 159 160 Lactate Dehydrogenase 161 Lactate Dehydrogenase • It is a tetramer of MW 14000 • It provides a good example of the occurrence of isoenzymes • There are five forms of the enzymes can be separated by electrophoresis • The different forms arise from five possible way of assembling a tetramer from two types of subunits (a4, a3b, a2b2, ab3 and b4) 162 Lacte Dehydrogenase Isoenzymes LD 1 LD 2 LD 3 LD 4 LD 5 163 Lactate Dehydrogenase Isoenzymes, Cont’d Heart 60 50 40 % 30 Distribution 20 LD-1 LD-2 LD-3 LD-4 LD-5 10 0 164 Lactate Dehydrogenase Isoenzymes, Cont’d Skeletal Muscle 45 40 35 30 25 % Distribution 20 15 10 5 0 LD-1 LD-2 LD-3 LD-4 LD-5 165 Molecular Structure of LDH LD 1 LD 2 LD 3 H H H H H H H H M H M M M M H M M M M M LD 5 LD4 166 LDH Isoenzymes in Liver 80 70 60 50 % 40 Distribution 30 20 10 0 LD 1 LD 2 LD 3 LD 4 LD 5 167 LDH Isoenzymes in Serum 40 35 30 25 % Total 20 Activity 15 10 LD-1 LD-2 LD-3 LD-4 & LD-5 5 0 168 169 LDH Assays Pyruvate O CH3 C COOH H+ + NADH Lactate OH CH3 CH COOH NAD 170 • The NAD (colored) is bound in a bent conformation: – Only part of the LDH enzyme is shown – The a-helices are displayed as bands, the bpleated sheets as arrows – Amino acid side chains that are in direct contact with NAD are outlined 171 NAD Binding Domain • (a) It consists of a 6stranded parallel bsheet and a 4 ahelix • (b) NAD binds in an extended conformation through H bonds and salt bridges (b) (a) 172 The tetramer of the M4 isoenzyme 173 Active Site of LDH • The active site of LDH showing the relative arrangement of reacting groups • The substrate pyruvate is shown; the -CH3 group is replaced by -NH2 to form oxamate • The hydride transfer is indicated by the bold arrow, hydrogen transfer by light arrow 174 Mechanism of Lactate Dehydrogease 175 Mechanism of Lactate Dehydrogease Arg-171 Arg -109 Hydride ion (H:-) is transferred from C-2 of lactate to the C-4 of NAD+ O His-195 H CH3 C O C N H O Lactate N H O B: NH2 Electron sink (Stored 2 electrons and one H+). Source & Where? + N R NAD+ 176 Lacate Dehydrogeanse O CH3C COO Pyruvate H H + .. O NH2 N R NADH His H N N BH+ 177 Ordered mechanism for lactate dehydrogenase • Reaction of lactate dehydrogenase • NAD+ is bound first and NADH released last 178 Alcohol Dehydrogenase 179 Alcohol Dehydrogenase • ADH is a homodimer • Each monomer has 374 residues with molecular weight of 74000 dalton • There are two domains: – The NAD+-binding domain (residues 176-318) consists of a central b-sheet of 6 strands flanked by a helices. NAD+ binds to the C-terminus of the b-sheet – The catalytic domain (residues 1-175, 319-374) also has a a/b structure 180 Alcohol Dehydrogenase • ADH binds two zinc ions: – One structural role – One catalytic role • There are two Zn2+ cations per monomer, one at the catalytic site being mandatory for catalysis • The catalytic zinc coordinates with two sulfur atoms from (3) Cys 46, Cys 174, and a nitrogen atom from His 67 • An ionizable water molecule occupies the fourth position on the zinc 181 Alcohol Dehydrogenase • The fifth and final zinc coordinate is the oxygen from the alcohol • In the active site there are three amino acids, Phe-93, Leu-57 and Leu-116, that work in concert to provide the three point binding of the alcohol substrate • This binding accounts for the stereospecific removal of the pro-R hydrogen 182 Alcohol Dehydrogenase 183 Alcohol Dehydrogenase 184 Alcohol Dehydrogenase ADH is a homodimer 185 Reaction of ADH 186 Dehydrogenase Stereospecificity 187 STEREOCENTERS One of the ways a molecule can be chiral is to have a stereocenter A stereocenter is an atom, or a group of atoms, that can potentially cause a molecule to be chiral stereocenters can give rise to chirality 188 STEREOGENIC CARBONS (called “chiral carbons” in older literature) Cl H stereocenter F Br A stereogenic carbon is tetrahedral and has four different groups attached 189 H F Cl Br plane of symmetry Cl Cl Br Cl Cl Cl Br Cl side view edge view 190 CONFIGURATION ABSOLUTE CONFIGURATION (R /S) 191 CONFIGURATION The three dimensional arrangement of the groups attached to an atom Stereoisomers differ in the configuration at one or more of their atoms 192 CONFIGURATION: relates to the three dimensional sense of attachment for groups attached to a chiral atom or group of atoms (i.e., attached to a stereocenter) clockwise 1 2 2 C counter clockwise C 4 4 3 view with substituent of lowest priority in back 1 R 3 (rectus) S (sinister) 193 DETERMINATION OF R/S CONFIGURATION IN FISCHER PROJECTIONS 194 PLACE THE PRIORITY = 4 GROUP IN ONE OF THE VERTICAL POSITIONS, THEN LOOK AT THE OTHER THREE 2 4 CHO 4 H OH 1 CH2OH OHC 2 CH2OH OH R 3 3 alternatively: 1 CHO 4 OH 1 CH2OH 3 BOTH IN BACK SAME RESULT 1 2 H #4 at top position H OH R 3 2 CHO HOCH2 4 H 195 #4 at bottom position FOR THE MENTALLY AGILE WHY BOTHER INTERCHANGING? JUST REVERSE YOUR RESULT! Same molecule as on previous slide. 2 CHO 4 H S OH 1 reverse R Same result as before. CH2OH 3 H coming toward you 196 THE SIMPLEST WAY OF ASSIGNING R/S CONFIGURATION WAS GIVEN BY EPLING (1982) 1. FIX THE PRIORITY 2. TRACE A SEMICIRCLE JOINING a IGNORING d b c 3. CLOCKWISE IS ‘R’ AND ANTICLOCKWISE ‘S’ IF ‘d’ IS VERTICAL (TOP OR BOTTOM) 4. IF ‘d’ IS ON THE HORIZONTAL LINE REVERSE THE NOTATION 197 Prochiral Center Ethanol Acetaldehyde 198 Prochiral Center NAD+ NADH 199 Alcohol Dehydrogenase: Pro-chirality R- 3 R Pro-S face H3C 2 O H3C 1 OH H 4 S H enantiomers Pro-R face R- 1 OH H 4 2 H3C R R 3 1 OH OH ethanol H3C 2 H3C R pro-R H hydrogen H pro-S hydrogen 3 D H 4 1 OH 2 H3C S H’s are enantiotopic, chemically equivalent 4 H D 3 200 201