淀粉酶PPT學習教案

1、會計學1淀粉酶淀粉酶第1頁/共125頁第2頁/共125頁第3頁/共125頁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頁/共125頁 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頁/共125頁第6頁/共125頁第7頁/共125頁第8頁/共125頁Pepsin crystals(X90)Northrop, J. H. (1930)“Crystallin pepsin, 1: Isolation and tests of purity” J. Gen . Physiol. 13:739-766.

3、第9頁/共125頁 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頁/共125頁Sidney Altman visiting PKU第11頁/共125頁第12頁/共125頁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頁/共125頁第14頁/共125頁xtraordinarily powerful;Highly specific;Be often regulated.第15頁/共125頁第16頁/共125頁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頁/共125頁Each enzyme has at least one active site第18頁/共125頁第19頁/共125頁Cosubstrates?第20頁/共125頁第21頁/共125頁(Vitamins)第22頁/共125頁est

7、ablished (e.g., pepsin, trypsin).第23頁/共125頁第24頁/共125頁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頁/共125頁La

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

10、 in oforDG 第30頁/共125頁is second order for a double substrate reaction (units can be M-1s-1). 第31頁/共125頁第32頁/共125頁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頁/共125頁第34頁/共125頁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頁/共125頁第3

12、6頁/共125頁0affected by S at high S.第37頁/共125頁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、頁/共125頁k 1k 2()第39頁/共125頁k 1k -1k 2Km is called the Michaelis constant.V0 = k2ES第40頁/共125頁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頁/共125頁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頁/共125頁第43頁/共125頁The double reciprocal plot:1/V0 vs 1/S第44頁/共125頁第45頁/共125頁k -1k 1第46頁/共125頁第47頁/共125頁converted to product in a given unit of time per enzyme molecule when the enzyme is saturated with substra

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

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

17、p distinguish these two mechanisms.第55頁/共125頁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頁/共125頁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頁/共125頁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頁/共125頁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頁/共125頁第60頁/共125頁“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頁/共125頁Stopped-flow apparatus forpre-steady state kinetics(since 1940s) Solutio

22、ns are forcedtogether very rapidly.第62頁/共125頁Quench flow apparatusfor rapid kinetics 第63頁/共125頁第64頁/共125頁ESEIE + PHow would the Km and Vmax be affected?KIEX第65頁/共125頁Apparent Vmax and Km values: Vmax unchanged, Km increases 第66頁/共125頁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)反競爭性抑制劑How would Km and Vmax be affected?EESESIE + PX第67頁/共125頁Both Vmax and Km decreases (but Vmax/Km unchanged).第68頁/共125頁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頁/共125頁Vmax decreases, Km increases.第70頁/共125頁第71頁/共125頁Irreversible inhibitorschemically modify or by the action of the specific enzym

25、e.第72頁/共125頁groups in the active sites of enzymes, thus providing an alternative reaction path.第73頁/共125頁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頁/共125頁An effective enzyme must have

26、its active sitecomplementary to thetransition state of the reaction.ES E-transition stateE + PActivation energy increases!第75頁/共125頁第76頁/共125頁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頁/共125頁第78頁/共125頁第79頁/共125頁第80頁/共125頁Many chemical reactionscan be promoted bygeneral acid-base catalysisTemporarily donating or accepting a proton.第81頁/共125頁Side chains of many amino acid residues c

28、an act as general acid-base第82頁/共125頁pH optimumpH optimun at 第83頁/共125頁Principles illustrated:Transition-state stabilization;General acid-base catalysis;Covalent catalysis.第84頁/共125頁第85頁/共125頁 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頁/共125頁Km = 20 mMKcat = 77 s-1Yellow productColorless substrateThis reaction is far slower than the hydrolysis of peptides!FastSlow第87頁/共125頁“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頁/共125頁第89頁/共125頁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頁/共125頁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頁/共125頁The hydrolysis ofa peptide bondat neutral pH without catalysiswill take 10-1000years!第92頁/共125頁第93頁/共125頁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頁/共125頁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頁/共125頁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頁/共125頁Water (the second substrate) then enters the active site.Entering ofS2Acyl-EE

36、S2第97頁/共125頁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頁/共125頁His57 acts as a general acidagain in breaking the c

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

38、uctEESAcyl-EES2EP2AcylationphaseDeacylationphaseThe proposed completecatalytic cycle of chymotrypsin(rate enhancement: 109)A Ping-Pong Mechanism第101頁/共125頁Val Val第102頁/共125頁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頁/共125頁第104頁/共125頁Ile is not a stericanalogue of the substrate of Thr dehydratase!第105頁/共125頁the amount of specific enzymes).第106頁/共125頁第107頁/共125頁第108頁/共125頁catalytic trimercatalytic t