生物化學(xué)：Chapter 6Enzyme catalysis

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1、Chapter 6Chapter 6 Enzyme catalysisBiochemistry Lecture (for Oct 18 and 23, 2012)Extreme efficiency of catalysis;High specificity on substrate and product;Regulation of activity in response to changes.What are enzymes?Enzymes affect reaction rates, not equilibria!Enzymes speed up the thermodynamical

2、ly favorable reactions to reach equilibria by lowering the activation energy (G), using the binding energy (GB).Enzymes catalyze the forward and backward reactions equally. a prosthetic group2 H2O2 2 H2O + O2 200,000 catalytic events/second/subunit (near the diffusion-controlled limit). The reaction

3、 is sped up by a billion fold!(tetramers)Fe3+ 1000 foldHemoglobin 1 ,000,000 foldCatalase 1 ,000,000,000 foldRate enhancementActive siteH2O2-induced oxidative damage was found to be a key element in senile hair graying (2009)! Enzymes can be small or large4-oxalocrotonate tautomeraseFatty acid synth

4、ase (human)Monomer: 62 residuesMonomer: 2504 residuesThe discovery of enzymes as the biocatalysts (1) Early studies: Understanding the nature of animal digestion (meat by stomach secretion and the conversion of starch to sugars by plant extracts and saliva) and fermentation (from sugar to alcohol).(

5、19th century) Diastase (later amylase淀粉酶): First enzyme discovered (in 1833) from geminating barley and saliva, liquefies starch paste and converted it into sugar.The discovery of enzymes as the biocatalysts (2) Pepsin: first discovered in 1834 as the active principle in the acid extract of gastric

6、mucosa causing the dissolution of coagulated egg white. Other “soluble ferments”. “Enzyme (something in yeast) ” was first coined for such “unorganized non-living ferments” by Khne in 1877. KhneTwo explanations were provided to explain the biological fermentation of sugar (19th century) Justus von L

7、iebig (Germany): fermentation caused by transmission of vibration from particles of ferment to particles of the fermenting material. Louis Pasteur (France): Fermentation a property inseparable from living cells.Liebig(1803-1873)PasteurSucrose was found to protect invertase activity (1890) OSULLIVAN,

8、 C. and TOMPSON, F. W. (1890) “Invertase: a contribution to the history of an enzyme or unorganized ferment” J. Chem. Soc. 57:834931. The activity of invertase in the presence of sucrose survives a temperature that completely destroys it if sucrose is absent. Enzyme combination with sucrose was sugg

9、ested.Early observations on the rate of fermentation by yeast (1892)BROWN, A. J. (1892) “Influence of oxygen and concentration on alcohol fermentation” J. Chem. Soc. 61, 369385.BROWN, A. J. (1902) “Enzyme action” J. Chem. Soc. 81, 373386. The rate of fermentation of sucrose in the presence of yeast

10、seemed to be independent of the amount of sucrose present, but on the amount of the enzyme. Invertase molecules present in the yeast was proposed to form addition complexes with sucrose.Grapes fermenting C6H12O6 2C2H5OH + 2CO2 The lock-and-key metaphor was proposed to explain the exquisite specifici

11、ty of enzymes (1894) FISCHER, E. (1894) “Einfluss der Configuration auf die Wirkung den Enzyme” Ber. Dtsch. Chem. Ges. 27:29852993. Invertin only hydrolyzes a a-methylglucoside. Emulsin hydrolyzes only b b-methylglucoside. These two enzymes must consist of “asymmetrically built molecules”.Emil Fisch

12、er(1852-1919)a a-methylglucosideCell-free extracts of yeast was reported to carry out sugar fermentation (1897) Fermentation is a chemical process, not a “vital process”. A “zymase” was proposed to be present in yeast cells and responsible for fermentation.A mathematical equation established to desc

13、ribe relationship between rate and substrate concentration (1913) Seeking an integrated form of the rate equations by studying invertase. Initial rate (v) of invertase-catalyzed reaction monitored at several sucrose concentrations. Theory: Invertase forms a complex with sucrose that is very labile a

14、nd decays to free enzyme, glucose and fructose. Theory: Rate must be proportional to concentration of sucrose-enzyme complex.Data fitting unveiled a constant to be calculated at the different substrate concentrationsC/KsThe constant (C/Ks) calculated being Vmax/Km, the specificity constant times the

15、 enzyme concentration (kcat/Km E0)!C = Vmax, is the total enzyme concentration, and k = KS, the dissociation constant of the sucrose-enzyme complex.For the first time, revealed a picture of the magnitude of the affinity of anenzyme for its substrate.A plotted version of the Michaelis-Menton dataJohn

16、son and Goody, (2011) The Original Michaelis Constant: Translation of the 1913 MichaelisMenten Paper, Biochemistry, 50:8264-8269 The products of the reaction were inhibitory.Current version of the Michaelis-Menten equation (Briggs & Haldane in 1925, using the steady state approximation) ES is in a s

17、teady-state and breakdown of ES is the slowest step.Leonor Michaelis(1875-1949)Leonora Menten(1879-1960)Km = (k2 + k-1)/k1Vmax = kcatE0J. B. S. Haldane (1892-1964)The actual meaning of Km depends on the reaction mechanism For If k2 is rate-limiting, k2k-1, then Km =k2/k1. If k2 and k-1 are comparabl

18、e, Km is a complex function of all three rate constants.k k -1k k 1Double reciprocal plots for determination of Km (Ks) introducedLineweaver & Burk (1934) The Determination of Enzyme Dissociation Constants, J. Ame. Chem.Soc. 56: 658666. Lineweaver noted the similarity between the MM equation and the

19、 Langmuir equation.(Ks-enzyme dissociation constant)The most cited paper of JACS, with more than 11000 citations. Hans Lineweaver(1907-2009)Dean Burk(1904-1988) Note: This equation can be used to describe a variety of bio-interactions,e.g, Mb-O2, Ab-Ag, DNA-DNA, protein-protein,etc.More reliable non

20、linear regression methods are used nowadays! Km is a constant for each substrate of an enzyme(Km: substrate concentration at half Vmax)Vmax is determined by kcat, the rate constant of the rate-limiting step Vmax = kcatEtkcat equals to k2 or k3 or a complex function of both, depending on which is the

21、 rate-limiting step.kcat is also called the turnover number: the number of substrate molecules converted to product in a given unit of time per enzyme molecule when the enzyme is saturated with substrate.40,000,000 molecules of H2O2 are convertedto H2O and O2 by one catalase molecule within one seco

22、nd!The kinetic parameters kcat and Km are often studied and compared for different enzymesKm often reflects the normal substrate concentration present in vivo for a certain enzyme. The catalytic efficiency of different enzymes is often compared by comparing their kcat/Km ratios (the specificity cons

23、tant). when SKmkcat/Km is an apparent second-order rate constant (with units of M-1S-1), relating the reaction rate to the concentrations of free enzyme and substrate.The value of kcat/Km has an upper limit (for the perfected enzymes) It can be no greater than k1.The decomposition of ES to E + P can

24、 occur no more frequently that E and S come together to form ES. The most efficient enzymes have kcat/Km values near the diffusion-controlled limit of 108 to 109 M-1S-1.Catalytic perfection (rate of reaction being diffusion-controlled) can be achieved by a combination of different values of kcat and

25、 Km.Rate enhancement is often used to describe the efficiency of an enzymeRate enhancement: ratio of the rates of conversion being catalyzed and the uncatalyzed reactions. k kununk kcatcatuncatalyzedcatalyzedNonenzymatic half-lifeUncatalyzed rate(kun, s-1) Catalyzed rate(kcat, s-1) Rate enhancement

26、(kcat/kun)EnzymeRate enhancement of selected enzymesThe unstable and elusive enzymes found to be proteins (1920s)Sumner, J. B. (1926) “ The isolation and crystallization of the enzyme urease” J. Biol. Chem. 69:435-441. Northrop, J. H. (1930) “Crystalline pepsin, 1: Isolation and tests of purity” J.

27、Gen. Physiol. 13:739-766. The crystals are purely protein as shown by all chemical tests. The proteins are the enzymes: reactivation; chemical tests.Octahedral crystals of jack bean ureasePepsin crystalsNobel Prize in 1946for his discovery that enzymes can be crystallized Northrop, J. H.Sumner, J. B

28、.Small heat stable chemicals were found to be needed for enzyme action (1906)Harden A, Young WJ (1906). The Alcoholic Ferment of Yeast-Juice, Part II: the Coferment of yeast juice, Proc. Roy. Soc. B: Biol. Sci. 78: 36975. Fermentation of glucose by yeast juice is dependent on a dialysable substance

29、which is not destroyed by heat. This heat stable factor was later identified as NAD. the first organic cofactor discovered.Arthur Harden(1865-1940)Hans von Euler-Chelpin(1873-1964) Nobel Prize 1929“for their investigations on the fermentation of sugar and fermentative enzymes”Non-protein chemical co

30、mpounds (organic or inorganic) found to be enzyme helpersProsthetic groups: tightly bound organic cofactors;Coenzymes: loosely bound organic cofactors (co-substrates).But there is no sharp division between them!Some organic cofactors are vitamins or are derived from vitamins. Many inorganic ions fou

31、nd to be cofactors of enzymesBeing the essential trace elements in nutrition.Enzymes categorized into 6 classes based on reactions catalyzed ( the nomenclature committee, IUBMB, 1961)Problem: uncontrolled naming of the rapidly increasing number of known enzymes. Some of the names in use were definit

32、ely misleading; others conveyed little or nothing about the nature of the reaction catalysed (e.g. catalase)http:/www.chem.qmul.ac.uk/iubmb/enzyme/The six classes of enzymes 1. OxidoreductasesOxidoreductases: transfer electrons from one substance (donor) to another (acceptor), e.g., dehydrogenases,

33、oxidases, oxygenases, reductases, peroxidases, and hydroxylases. 2.TransferasesTransferases: Transfer groups (amino, carboxyl, carbonyl, methyl, phosphoryl, and acyl) from one molecule to another, e.g., transaminases, transcarboxylases, and transmethylases.22 subclasses9 subclassesThe six classes of

34、 enzymes 3. HydrolasesHydrolases: Cleavage of bonds by adding water, e.g., lipases, phosphatases, and peptidases. 4. LyasesLyases: breaking of chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond, e.g., decaboxylases, hydratases, dehydratases, deaminases and s

35、ynthases.7 subclasses13 subclassesThe six classes of enzymes 5. IsomeasesIsomeases: structural rearrangement of isomers, e.g., racemases, epimerases, mutases and isomerases. 6. LigasesLigases: Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of A

36、TP, e.g. synthetases, carboyxlases and ligases.6 subclasses6 subclassesThe number of enzymes revealed have been increasing As reported by the Enzyme Commision or Enzyme Nomenclature committee of IUBMB:Year number of enzymes1961 712 1964 875 1972 1770 1978 2122 1984 2477 1992 3196Each enzyme possess

37、a conventional name, a Each enzyme possess a conventional name, a systematic name, and a EC number (assigned by systematic name, and a EC number (assigned by Enzyme Commission of IUBMBEnzyme Commission of IUBMBLactate dehydrogenase (lactate:NAD+ oxidoreductase)Lactate + NAD+ pyruvate + NADH + H+ 1 1

38、Type of electron donor (OH)Type of electron Acceptor (NAD) Enzymes can be reversibly suppressed by noncovalent binding of inhibitorsCompetitive Non-competitive Substrate & inhibitor compete for access to the enzymes active site;Sufficiently high concentrations of substrate out-compete the inhibitor!

39、Kmapp increases as it takes higher concentration of substrate to reach 1/2Vmax Substrate and inhibitor bind independentlybut inhibitor binding completely preventcatalysis.Binds to E, but not to ES Identical affinities for E and ES Substrate and product Inhibition might occur.Many drug molecules are

40、reversible inhibitors of key enzymesRitonavir (Norvir) is an inhibitor of the HIV protease.(for treating AIDS) High specificity and potency meansfew side effects and low toxicity. Sildenafil (Viagra) is an inhibitor of cGMP-specific phosphodiesterase type 5 (for treating male erectile dysfunction) M

41、ethotrexate,an inhibitor of dihydrofolate reductase,inhibits the synthesis of thymidine and purines.(for treating cancer) Chemical reactions hypothesized to occur via transition states Transition state: A high energy configuration of the reactants along the reaction coordinate. (Eyring and Polanyi,

42、1935). Being not a chemical species of any significant stability, but a fleeting molecular moment in which bonds are both broken and formed.Henry Eyring Michael PolanyiLifetimes 10-13 seconds The active site of enzymes was proposed to complement the strained configuration of the substrates (1946)Pau

43、ling, L. (1946) Molecular Architecture and Biological Reactions, Chem. Eng. News, 24:1375-1377. Pauling, L. (1948) Nature of forces between large molecules of biological interest, Nature 161:707-709. Active region of surface of enzyme has a configuration NOT complementary to the substrate in its nor

44、mal configuration, but rather in the strained configuration, or “activated complex”, of the reaction catalyzed, thus decreasing the activation energy and increase the rate of the reaction. If enzyme completely complementary in structure to substrate then no other molecule would compete successfully

45、with the substrate in combining with the enzyme, similar in behavior to antibodies; but an enzyme complementary to a strained substrate molecule would attract more strongly to itself a molecule resembling the strained substrate molecule than it would the substrate molecule.Pauling(1901-1994)Enzyme a

46、ctive site has to complement the transition state, NOT the substrateActivation energy will beIncreased, instead of decreased!which will decreasing the rate!ONLY this will work!Transition state analogs found to tightly bind to enzymes (1972)Secemski et al. (1972) A transition state analog for lysozym

47、e. J Biol Chem. 247:4740-8. Transition state analogs found to bind to enzymes 102 to 106 times more tightly than substrates. Substrates often participate several enzyme reactions, whereas the transition state tends to be characteristic of one particular enzyme. LactoneAlkoxyl carboniumAssumed transi

48、tion statefor the lysozyme-catalyzedreactionTransition stateanalog designedCatalytic antibodies were predicted by William Jencks (1969) If complementarity between the active site and the transition state contributes significantly to enzymatic catalysis, it should be possible to synthesize an enzyme

49、by constructing such an active site. One way to do this is to prepare an antibody to a haptenic group which resembles the transition state of a given reaction. The combining sites of such antibodies should be complementary to the transition state and should cause an acceleration by forcing bound sub

50、strates to resemble the transition state. William p. Jencks, Catalysis in Chemistry and Enzymology, 1969, p.288Catalytic antibodies (abzyme) were proved (1986) by Schultzs labTramontano et al. (1986) Catalytic antibodies, Science, 234:1566.Pollack et al. (1986) Selective chemical catalysis by an ant

51、ibody, Science, 234:1570.Postulated transition state (metallopeptidase)Transition state analog (as hapten)Substrate of theCatalytic antibodyPeter SchultzIrreversible inhibitors were used to identify key residues in the active site of chymotrypsin (1955, 1964)Turba and Gundlach,(1955) Amino acid sequ

52、ence in the area of the reactive serine group of the chymotrypsin molecule. Biochem Z. 327:186-8. Ong et al. (1964) The identification of the histidine residue at the active center of chymotrypsin. J Biol Chem. 240:694-8. Modification followed by sequencing: specific Ser-195 and His-57 residues assu

53、med to be in the active sites due to their high reactivity.The inhibitor, TPCK, is 14C-labeled.Only one of the 2 His residues, one out of the 25 Ser residues being labeled!Diisopropylphosphofluoridate (DIPF) Ser-195ChymotrypsinProteolytic enzymes found often exist as inactive zymogen precursors (193

54、3)Kunitz & Northrop JH. (1933) Isolation of a crystalline protein from pancreas and its conversion into a new crystlline proteolytic enzyme by trypsin. Science. 78:558-9.Kunitz M, Northrop JH. (1934) The isolation of crystalline trypsinogen and its conversion into crystalline trypsin. Science. 80:50

55、5-6 A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the active site) for it to become an active enzyme. The tertiary structure of the first enzyme (lysozyme) determined (1965)Blake et al. (1965) Structure of he

56、n egg-white lysozyme. A three-dimensional Fourier synthesis at 2 A resolution. Nature. 206:757-61. Johnson LN and Phillips DC. (1965). Structure of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 A resolution. Nature. 206:761-3. Action mechanism can be proposed based

57、on the enzyme-inhibitor complex structure.Lysozyme cleaves the polysaccharideof the bacterial cell wall.Structure of NAD-dependent lactate dehydrogenase determined (1970)Adams et al. (1970) Structure of lactate dehydrogenase at 2.8 A resolution. Nature. 227:1098-103. Rossmann et al. (1974) Chemical

58、and biological evolution of nucleotide-binding protein. Nature. 250:194-9. A common nucleotide binding motif (“Rossmann fold”) was found in all NAD- and FAD-dependent enzymes.Rossmann foldMichael RossmannPyruvate lactateChymotrypsin catalysis was found to occur in two stages (1954) Hartley and Kilby

59、, (1954) The Reaction of p-Nitrophenyl Esters with Chymotrypsin and Insulin, Biochem. J.,56:288-297. Ester was used as substrate to make the reaction slower. The catalysis appears to occur in two stages: a rapid acetylation, releasing one p-nitrophenol per enzyme, followed by a slow step.The slow li

60、near hydrolysis precededby a rapid initial reaction.The extrapolated linear hydrolysis plot did not passthrough the origin at zero time:Km = 20 mMKcat = 77 s-1Colorless substrateYellow productThis reaction is far slower than the hydrolysis of peptides!“burst” (fast) phase (rapid acylation of all Enz

61、ymes leading to release of p-nitrophenol)Slow phase (enzymes will be able to act again only after a slow deacylation step)The catalysis of chymotrypsinis biphasic as revealed by pre-steady state kinetics(burst kinetics)Milliseconds after mixingReflecting a single turnover of the enzyme Chymotrypsin

62、operates through a ping-pong mechanism Rates of individual steps for an enzyme-catalyzed reaction may be obtained by pre-steady state kinetics The enzyme (of large amount) is used in substrate quantities and the events on the enzyme are directly observed. Rates of many reaction steps may be measured

63、 independently. Very rapid mixing and sampling techniques are required (the enzyme and substrate have to be brought together in milliseconds and measurements also be made within short period of time).“Rapid kinetics” or “pre-steady-state kinetics”is applied to the observation of rates of systems tha

64、t occur in very short time intervals (usually ms or sub-ms scale ) and very low product concentrations. This period covers the time from the enzyme encountering its target (either a substrate, inhibitor or some other ligands) to the point of system settling to equilibrium. The concentration of ES wi

65、ll rise from zero to its steady-state value. (ms or sub-ms)Stopped-flow apparatus forpre-steady state kinetics(since 1940s) Solutions are forcedtogether very rapidly.Rapid mixingRapid samplingQuench flow apparatusfor rapid kinetics Rapid mixingRapid stoppingDetermination of the crystal structure of

66、chymotrypsin (1967) revealed a catalytic triad: Ser195, His57, Asp102.Matthews et al. (1967) Three-dimensional structure of tosyl-alpha-chymotrypsin. Nature. 214:652-6. Blow et al. (1969) Role of a buried acid group in the mechanism of action of chymotrypsin. Nature. 221:337-40.Chymotrypsin: three polypeptide chains linked by multiple disulfide bonds; a catalytic triad.His57Asp102Ser195Cleft for binding extended substratesTrypsin, sharing a 40% identity withchymotrypsin, has a very similar struc