生物化学原理课件（英文）：Chapter10 Mechanisms of enzyme catalysis

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1、Chapter 10 Mechanisms of Enzyme CatalysisOutlineMajor methods for studying enzyme mechanisms“The transition-state stabilization” theoryMechanisms of transition state stabilization 1.Proximity and Orientation2.General Acid-base catalysis3.Electrostatic catalysis4.Metal catalysis5.Covalent catalysis6.

2、Substrate strainStructure and function of several common enzymes1.Proteases2.Lysozyme (self-study)Major methods for studying enzyme mechanisms1.Find out the conservative residues by aligning homologous enzymes2.Study the 3-D structure of enzymes3.Perform site-directed mutagenesis4.Carry out the kine

3、tic analysis5.Make chemical modification6.Molecular Computer Mimics Enzymes bring substrates together in the proper orientation for a reaction to occur Enzymes possess functional groups that stabilize the transition state of the reactionEEnzymes lower the activation Gibbs energy of a reaction Enzyme

4、s drive thermodynamically unfavorable reactions by coupling them to thermodynamically favorable reactionsHow do enzymes work?An imaginary stickase designed to catalyze breakage of a metal stickEvidences of stabilizing the transition state of the reaction Transition analogues are potent inhibitors Ab

5、zymesEnzymes have evolved to recognize the transition state of the reaction they catalyzeTo design an enzyme inhibitor, we should try to mimic the transition state of the reaction, not the substrates or productsDrug design: Transition state analoguesRiboseNCNCHCHNH2OCytidineUridineCNCHCHOOHNRibosecy

6、tidinedeaminaseCytidine deaminase is a target for anticancer drugsAn example - tetrahydrouridine is a transition state analogue of cytidine deaminase Antibodies are immunoglobulins. Antibodies are elicited in an organizm in response to immunological challenge by a foreign molecule called antigens; A

7、ntibodies elicited in response to transition state analogs have the ability to stabilize the transition state and thus can catalyze a reaction by forcing the substrate into the transition state structure; Examples of abzymes:Abzymes - Catalytic AntibodiesImmunize animals, produce antibodyHydrolysize

8、abzymeCocainePreparation of abzyme catalyzing hydrolysis of cocaineHow do enzymes stabilize the transition state of a reaction1.Catalysis by proximity and orientation2.General Acid-base catalysis3. Electrostatic catalysis4.Metal catalysis5.Covalent catalysis6.Substrate strainCatalysis by proximity a

9、nd orientation This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together. This reduces the entropy of the reactants and thus makes reactions such as ligations or addition reactions more favorable, there is a reduction in the

10、overall loss of entropy when two reactants become a single product. This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzyme gives the reaction intramolecular character, which gives a massive rate increaseIntramolecular and intermo

11、lecular reactionsGeneral acid catalysis is a process in which proton transfer from an acid lowers the free energy of a reactions transition state.A reaction may also be stimulated by general base catalysis if its rate is increased by proton abstraction by a base .Some reactions may be simultaneously

12、 subject to both processes; these are concerted acidbase catalyzed reactions.General Acid-base catalysisGeneral base catalysisGeneral Acid catalysisGeneral Acid-base catalysisMechanisms of ketoenol tautomerization. (a) Uncatalyzed. (b) General acid catalyzed. (c) General base catalyzed.Differences b

13、etween Specific acid/base catalysis and general acid/base catalysisThe RNase A mechanism (1)The RNase A mechanism (2)OCH3COOCH3CO-CCH3OOCH3COO-SlowEnzymeFastOCH3COOCH3CO-CCH3OOCH3COOEnzymeHOCCH2OHGeneral Acid-Base CatalysisLysozyme is an example of general acid catalysisGlu 35 in Lysozyme is surroun

14、ded by hydrophobic amino acidsEnzymes use charged amino acids to neutralize charges that develop during formation of the transition state of a reaction. This is known as electrostatic catalysis.Electrostatic catalysisConsider the hydrolysis of a peptide bondWhy does neutralizing the charge that deve

15、lops in the transition state increase the reaction rate?Lysozyme uses electrostatic catalysis to catalyze the cleavage of polysaccharidesOOHCGlu 35OOAsp 52-CNHCOOCH2OHCH3OHHHOHBOCH2OHNHOHHOHCOCH3CHCH3COO-AO-OOCGlu 35OOAsp 52-CNHCOOCH2OHCH3OHHHOHBOCH2OHNHOHHOHCOCH3CHCH3COO-AOH+-OOCGlu 35OOAsp 52-CNHC

16、OOCH2OHCH3OHHHOHBOH+OCH2OHNHOHHOHCOCH3CHCH3COO-A-OOCGlu 35OOAsp 52-CNHCOOCH2OHCH3OHHHOHBOH+OCH2OHNHOHHOHCOCH3CHCH3COO-AHOHOOHCGlu 35OOAsp 52-CNHCOOCH2OHCH3OHHHOHBOHOCH2OHNHOHHOHCOCH3CHCH3COO-AOHThe pH profile of polysaccharide hydrolysis reflects the pKa values of Glu 35 and Asp 52 Metals can act as

17、 Lewis acids Metals can stabilize charges that develop in the transition state Metals can accept and donate electrons in oxidation-reduction reactions Metals can be important for the structure of the enzymeMetal catalysis A Lewis acid is a species that can accept a pair of electrons for bondingZn2 +

18、 + H2O ZnOH + H+Zn functions as a Lewis acid in carbonic anhydraseMetals can act as Lewis acidsZn functions as a Lewis acid in carbonic anhydrasecarbonicanhydraseZn2+OHH.CO2carbonicanhydraseZn+OH-H+OCO.carbonicanhydraseZn2+OHCOOHMetals can stabilize charges that develop in the transition stateMetals

19、 can accept and donate electrons in oxidation-reduction reactionsThis is the principal effect of induced fit binding. This induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the activation energy and helping

20、catalyze the reaction. In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate residues in the active site.Substrate strainLysozyme is an example of how enzymes create strain in the substrateNHHHO CCHOOHCCH3COO-OHHNHHHCCOOHOCCH3COO-OHOCCH3COO

21、-OHHNHHHCC+H General acid catalysis Glu 35 donates a proton Electrostatic catalysis the positively charged carbonium ion is stabilized by Asp 52 Strained substrate conformation the sugar bound to the active site on the enzyme is forced to adopt a half chair conformationLysozyme catalyzed cleavage of

22、 polysacharrides involves: Covalent catalysis accelerates reaction rates through the transient formation of a catalystsubstrate covalent bond. Usually, this covalent bond is formed by the reaction of a nucleophilic group on the catalyst with an electrophilic group on the substrate, and hence this fo

23、rm of catalysis is often also called nucleophilic catalysis. In addition, several coenzymes, notably thiamine pyrophosphate and pyridoxal phosphate, function in association with their apoenzymes as covalent catalysts.Covalent catalysisIn covalent catalysis, the enzyme forms a transient covalent inte

24、rmediate with the substrates of the reaction.Covalent catalysisBiologically important nucleophilic and electrophilic groupsBiologically important nucleophilic and electrophilic groupsThe decarboxylation of acetoacetateThe uncatalyzed reaction mechanism is at the top, and the mechanism as catalyzed b

25、y primary amines is at the bottom-Catalyze group transfer to waterUncatalyzed hydrolysis of a peptide linkage is very slow with a half-life at neutral pH and 25 oC of 300-600 years What are Proteases?1.Serine proteases - inactivated by diisopropyl fluorophosphate2.Metalloproteases - inactivated by E

26、DTA or o-phenanthroline (example - thermolysin)3.Carboxyl proteases - not active at neutral or slightly alkaline pH (example - HIV protease)4.Thiol proteases - inactivated by iodoacetamide or iodoacetateFour Major Classes of ProteasesGeneral Characteristics of Proteases1.They all go through a transi

27、tion state in which the normally trigonal carbonyl carbon of the peptide bond becomes tetrahedral.2.This tetrahedral geometry occurs because of the temporary addition of a nucleophile.3.In the case of the serine and thiol proteases, the nucleophile is the serine hydroxyl or cysteine thiol at the act

28、ive site whereas in the other two classes it is a water molecule.4.The reaction proceeds more directly in the metalloproteases and the carboxyl proteases, whereas the acyl-enzyme intermediate that occurs with the serine and thiol proteases must be hydrolyzed by water in a second step. 1.Best underst

29、ood of the proteases 2.All have a peculiarly reactive Ser residue3.There are three distinct structural families - they have very similar mechanisms of action but are not detectably related - they provide one of the most striking examples of apparent evolutionary convergence 4.One family is represent

30、ed by the bacterial protease subtilisin, another by enzymes including chymotrypsin, trypsin, elastase, thrombin, kallikrein, etc. and, finally a family represented by wheat germ serine carboxypeptidase II 5.Members in the trypsin family are involved in a range of cellular functions including blood c

31、lotting, complement activation, hormone production, and fertilization Serine Proteases1.all share an oxyanion hole2.all contain a catalytic triad - Serine, Histidine, and Aspartate: His directly abstracts a proton from Ser thereby converting it to an alkoxide ion3.They differ most strikingly in thei

32、r preference for amino acid side chains on the C-side of Peptide bond4.Trypsin cleaves bonds only after Lys and Arg residues, chymotrypsin after large hydrophobic residues; the other proteases of this family have less distinct preferences5.Proteases can catalyze hydrolysis of both amides and estersG

33、eneral Characteristics of Serine Proteases1. The serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the scissile peptide bond of the substrate.2. A pair of electrons on the histidine nitrogen has the ability to accept the hydrogen from the serine -OH group

34、, thus coordinating the attack of the peptide bond.3. The carboxyl group on the aspartic acid in turn hydrogen bonds with the histidine, making the nitrogen atom mentioned above much more electronegative.Catalytic Triad of Serine ProteasesThe different substrate specificities of the serine proteases

35、 are due to differences in their specificity pocketsThe catalytic mechanism of the serine proteasesThe catalytic mechanism of the serine proteasesThe catalytic mechanism of the serine proteasesThe catalytic mechanism of the serine proteasesThe catalytic mechanism of the serine proteasesThe catalytic mechanism of the serine proteasesOxyanion hole helps to stabilize transition state in the serine proteases