淀粉酶PPT学习教案

1、会计学1淀粉酶淀粉酶第1页/共125页第2页/共125页第3页/共125页John Burdon Sanderson Haldane (1892-1964) Leonor Michaelis(1875-1949)Maud Menten(1879-1960)A GermanA CanadianA BritishGeneticistBefore it was known that enzymes are proteins!第4页/共125页 The Nobel Prize in Chemistry 1907for his biochemical researches and his discove

2、ry of cell-free fermentation Eduard Buchner Germany Landwirtschaftliche Hochschule (Agricultural College) Berlin, Germany b. 1860, d. 1917.第5页/共125页第6页/共125页第7页/共125页第8页/共125页Pepsin crystals(X90)Northrop, J. H. (1930)“Crystallin pepsin, 1: Isolation and tests of purity” J. Gen . Physiol. 13:739-766.

3、第9页/共125页 The Nobel Prize in Chemistry 1946“ for his discovery that enzymes can be crystallizedfor their preparation of enzymes and virus proteins in a pure form James Batcheller Sumner John Howard Northrop Wendell Meredith Stanley 1/2 of the prize 1/4 of the prize 1/4 of the prize Cornell Universit

4、y Ithaca, NY, USARockefeller Institute for Medical Research Princeton, NJ, USA Rockefeller Institute for Medical Research Princeton, NJ, USA 1887-1955 1891-1987 1904-1971第10页/共125页Sidney Altman visiting PKU第11页/共125页第12页/共125页The rate is proportional to the concentration of the reactantin a typical

5、chemical reaction.Enzymes however showed a saturation kinetics: formation of ES complex was hypothesized (1902).第13页/共125页第14页/共125页xtraordinarily powerful;Highly specific;Be often regulated.第15页/共125页第16页/共125页a prosthetic group2 H2O2 2 H2O + O2 200,000 catalytic events/second/subunit (near the dif

6、fusion-controlled limit). The reaction is sped up by a billion fold!(tetramers)Fe3+ 1000 foldHemoglobin 1 ,000,000 foldCatalase 1 ,000,000,000 foldRate enhancementActive site第17页/共125页Each enzyme has at least one active site第18页/共125页第19页/共125页Cosubstrates?第20页/共125页第21页/共125页(Vitamins)第22页/共125页est

7、ablished (e.g., pepsin, trypsin).第23页/共125页第24页/共125页Transfer electrons (hydride ions or H atoms);play a major role in energy metabolism.the transfer of functional groups to water.e.g., the transfer of a phosphoryl group from ATP to many different acceptors.These are direct bond breaking reactions w

8、ithout being attacked by another reactant such as H2O. In chemical terms, they would be described aselimination and addition reactions.Leading to the formation of C-C, C-S,C-O, C-N bonds.Lactate dehydrogenaseNMP kinaseChymotrypsinFumaraseTriose phosphateisomeraseAminoacyl-tRNA synthetase第25页/共125页La

9、ctate dehydrogenase (lactate:NAD+ oxidoreductase)lactate + NAD+ pyruvate + NADH + H+ 1Indicatestype of substrateIndicatestype ofcofactor 第26页/共125页第27页/共125页第28页/共125页determined at . n The value of K eq is determined by the standard free energy changeof the reaction.第29页/共125页For each 10-fold change

10、 in oforDG 第30页/共125页is second order for a double substrate reaction (units can be M-1s-1). 第31页/共125页第32页/共125页The rate of a reaction is determined by the value of activation energy (G ) kT hThe relationship between k andG is inverse and exponential! G A lower activation energy means a faster react

11、ion rate.第33页/共125页第34页/共125页An enzyme provides an alternative pathway for the conversion of the substrates to the products, thuslowers the activation energy and speeds up the reaction.Enzymes make the rate constantslarger and only catalyze reactions that are thermodynamically favorable.第35页/共125页第3

12、6页/共125页0affected by S at high S.第37页/共125页Hyperbolic relationship between V0 and S(similar to the O2 binding curve of myoglobin)The effect on V0 of varying S is measured when the enzyme concentration is held constant.Vmax is extrapolated from the plot: V0 approaches but never quite reaches Vmax.第38

13、页/共125页k 1k 2()第39页/共125页k 1k -1k 2Km is called the Michaelis constant.V0 = k2ES第40页/共125页The maximum velocity is achieved when all the enzyme is saturated by substrate, i.e., when ES =Et. Thus Vmax =k2EtThe Michaelis-Menten Equation第41页/共125页The Michaelis-Menten Equation nicelydescribes the experim

14、ental observations.When S KmThe substrate concentration at which V0 is half maximal is Km第42页/共125页第43页/共125页The double reciprocal plot:1/V0 vs 1/S第44页/共125页第45页/共125页k -1k 1第46页/共125页第47页/共125页converted to product in a given unit of time per enzyme molecule when the enzyme is saturated with substra

15、te.第48页/共125页40,000,000 molecules of H2O2 are convertedto H2O and O2 by one catalase molecule within one second!第49页/共125页第50页/共125页第51页/共125页Catalytic perfection (rate of reaction being diffusion-controlled) can be achieved by a combination of different values of kcat and Km.第52页/共125页Rate enhancem

16、ent: ratio of the rates of the catalyzed and the uncatalyzed reactions. kcatkcatcatalyzeduncatalyzed第53页/共125页Nonenzymatic half-lifeUncatalyzed rate(kun, s-1) Catalyzed rate(kcat, s-1) Rate enhancement (kcat/kun)EnzymeRate enhancement by selected enzymes第54页/共125页nSteady-state kinetics can often hel

17、p distinguish these two mechanisms.第55页/共125页In those enzyme-catalyzed bisubstrate reactions where aternary complex is formed, the two substrates may either bind in a random sequence or in a specific order.第56页/共125页Maintaining the concentrationof one substrate (S2) constant,the double reciprocal pl

18、ots made by varying the concentrationof the other substrate (S1) willintersect.For those reactionswhere ternary complexis formed:第57页/共125页No ternary complex is formed in the Ping-Pong (or double displacement) mechanism: The first substrate is converted to a product that leaves the enzyme active sit

19、e before the second substrate enters.第58页/共125页For enzymes having Ping-Pong mechanisms (ternary complex not formed).Maintaining the concentrationof one substrate (S2) constant,the double reciprocal plots made by varying the concentrationof the other substrate (S1) will notintersect.As S2 increases,

20、Vmax increases,as does the Km for S1.S1第59页/共125页第60页/共125页“Rapid kinetics” or “pre-steady-state kinetics”is applied to the observation of rates of systems that occur in very short time intervals (usually ms or sub-ms scale ) and very low product concentrations. This period covers the time from the

21、enzyme encountering its target (either a substrate, inhibitor or some other ligands) to the point of system settling to equilibrium. The concentration of ES will rise from zero to its steady-state value. (ms or sub-ms)第61页/共125页Stopped-flow apparatus forpre-steady state kinetics(since 1940s) Solutio

22、ns are forcedtogether very rapidly.第62页/共125页Quench flow apparatusfor rapid kinetics 第63页/共125页第64页/共125页ESEIE + PHow would the Km and Vmax be affected?KIEX第65页/共125页Apparent Vmax and Km values: Vmax unchanged, Km increases 第66页/共125页Uncompetitive inhibitors are present only for enzymes catalyzing r

23、eactions of two or more substrates (with ordered substrate binding): analogs of S2 will act as uncompetitive inhibitor for the enzyme (relative to S1)反竞争性抑制剂How would Km and Vmax be affected?EESESIE + PX第67页/共125页Both Vmax and Km decreases (but Vmax/Km unchanged).第68页/共125页Mixed inhibitors are prese

24、nt for enzymesof random ordered substrate binding.EESEIESIE + PXNoncompetitive inhibitor: binding of I does not affect binding of S; Vmax decreases, Km unchanged.第69页/共125页Vmax decreases, Km increases.第70页/共125页第71页/共125页Irreversible inhibitorschemically modify or by the action of the specific enzym

25、e.第72页/共125页groups in the active sites of enzymes, thus providing an alternative reaction path.第73页/共125页nAccording to the transition state theory, an enzyme must be complementary to the reaction transition state of the reactant (Haldane, 1930; Pauling, 1946).第74页/共125页An effective enzyme must have

26、its active sitecomplementary to thetransition state of the reaction.ES E-transition stateE + PActivation energy increases!第75页/共125页第76页/共125页Transition-state analogs can be designed according to theproposed reaction mechanism and used as antigens for making catalytic antibodies.Transition-state ana

27、logTransition-state analogTransition-stateTransition-stateCatalytic antibodiesCatalytic antibodies第77页/共125页第78页/共125页第79页/共125页第80页/共125页Many chemical reactionscan be promoted bygeneral acid-base catalysisTemporarily donating or accepting a proton.第81页/共125页Side chains of many amino acid residues c

28、an act as general acid-base第82页/共125页pH optimumpH optimun at 第83页/共125页Principles illustrated:Transition-state stabilization;General acid-base catalysis;Covalent catalysis.第84页/共125页第85页/共125页 TPCK alkylates His 57 Inactivation can be inhibited by b-phenylpropionate (competitive inhibitor) TPCK modi

29、fication does not occur when chymotrypsin is denatured in urea.第86页/共125页Km = 20 mMKcat = 77 s-1Yellow productColorless substrateThis reaction is far slower than the hydrolysis of peptides!FastSlow第87页/共125页“burst” (fast) phase (rapid acylation of all Enzymes leading to release of p-nitrophenol)Slow

30、 phase (enzymes will beable to act again only after a slow deacylation step)The catalysis of chymotrypsinis biphasic as revealed by pre-steady state kineticsMilliseconds after mixing第88页/共125页第89页/共125页Chymotrypsin: three polypeptide chains linked by multiple disulfide bonds; a catalytic triad.His57

31、Asp102Ser195Cleft for binding extended substratesTrypsin, sharing a 40% identity withchymotrypsin, has a very similar structure.Active site第90页/共125页A catalytic triad has been found in all serine proteases: the Ser is thus converted into a potent nucleophile (subtilisin has no homology with other Se

32、r protease members, but has the triad)第91页/共125页The hydrolysis ofa peptide bondat neutral pH without catalysiswill take 10-1000years!第92页/共125页第93页/共125页The peptide bond to be cleaved is positioned by the binding of the side chain of an adjacent hydrophobic residue in a special hydrophobic pocket.As

33、p102 functions only to orient His57.Formation of the ES complexESES1Formation of ES1第94页/共125页His57 acts as a general base indeprotonating Ser195, the alkoxideion then acts as a nucleophile, attacking the carbonyl carbon.Ser195 forms a covalent bond with the peptide (acylation) to be cleaved. a trig

34、onal C is turned into a tetrahedral C.The tetrahedral oxyanion intermediate is stabilized by the NHs of Gly193 and Ser195Preferential binding of the transition state: oxyanion hole stabilization of the negatively charged tetrahedral intermediate of the transition state.Pre-acylationES1oxyanion hole第

35、95页/共125页The amine product is then released from theactive site with the formation of an acyl-enzyme covalent intermediate.His57 acts as a general acidin cleaving the peptide bond.AcylationReleasing of P1ES1Acyl-E第96页/共125页Water (the second substrate) then enters the active site.Entering ofS2Acyl-EE

36、S2第97页/共125页His57 acts as a general base again, allowing water to attack the acyl-enzyme intermediate,forming another tetrahedraloxyanion intermediate, again stabilized by the NHs of Gly193 and Ser195 (similar to step 2) Pre-deacylationES2第98页/共125页His57 acts as a general acidagain in breaking the c

37、ovalentbond between the enzymeand substrate (deacylation) (similar to Step 3).DeacylationEP2第99页/共125页The second product(an acid) is released from the active site, with the enzyme recoveredto its original state.Release of P2Recovered enzymeEP2E第100页/共125页1st substrate1st product2nd substrate2nd prod

38、uctEESAcyl-EES2EP2AcylationphaseDeacylationphaseThe proposed completecatalytic cycle of chymotrypsin(rate enhancement: 109)A Ping-Pong Mechanism第101页/共125页Val Val第102页/共125页A dynamic process for chymotrypsin catalysis:A Ping Pang mechanism.Importance of theresidues wasexmined bysite-directed mutagenesis:The Ser and His residues are far more important than the Asp residue!第103页/共125页第104页/共125页Ile is not a stericanalogue of the substrate of Thr dehydratase!第105页/共125页the amount of specific enzymes).第106页/共125页第107页/共125页第108页/共125页catalytic trimercatalytic t