5.初級(jí)版課件4english vesion英文版-abridgedof enzyme catalysis

1、Chapter 10 Mechanisms of Enzyme CatalysisOutlineMajor methods for studying enzyme mechanisms“The transition-state stabilization” theoryMechanisms of transition state stabilization Proximity and OrientationGeneral Acid-base catalysisElectrostatic catalysisMetal catalysisCovalent catalysisSubstrate st

2、rainStructure and function of several common enzymesProteasesLysozyme (self-study)Major methods for studying enzyme mechanismsFind out the conservative residues by aligning homologous enzymesStudy the 3-D structure of enzymesPerform site-directed mutagenesisCarry out the kinetic analysisMake chemica

3、l modificationMolecular Computer Mimics Enzymes bring substrates together in the proper orientation for a reaction to occurEnzymes possess functional groups that stabilize the transition state of the reactionEnzymes lower the activation Gibbs energy of a reactionEnzymes drive thermodynamically unfav

4、orable 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 reactionTransition analogues are potent inhibitorsAbzymes Antibodies are immunoglobul

5、ins. Antibodies are elicited in an organizm in response to immunological challenge by a foreign molecule called antigens;Antibodies 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 t

6、ransition state structure;Examples of abzymes:Abzymes - Catalytic AntibodiesImmunize animals, produce antibodyHydrolysizeScreenabzymeCocainePreparation of abzyme catalyzing hydrolysis of cocaineHow do enzymes stabilize the transition state of a reactionCatalysis by proximity and orientationGeneral A

7、cid-base catalysis Electrostatic catalysisMetal catalysisCovalent catalysisSubstrate strainCatalysis by proximity and orientationThis 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 react

8、ants and thus makes reactions such as ligations or addition reactions more favorable, there is a reduction in the overall loss of entropy when two reactants e a single product.This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzym

9、e gives the reaction intramolecular character, which gives a massive rate increaseGeneral 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 p

10、roton abstraction by a base .Some reactions may be simultaneously subject to both processes; these are concerted acidbase catalyzed reactions.General Acid-base catalysisGeneral base catalysisGeneral Acid catalysisGeneral Acid-base catalysisLysozyme is an example of general acid catalysisGlu 35 in Ly

11、sozyme is surrounded 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

12、 charge that develops in the transition state increase the reaction rate?Lysozyme uses electrostatic catalysis to catalyze the cleavage of polysaccharidesMetals can act as Lewis acidsMetals can stabilize charges that develop in the transition stateMetals can accept and donate electrons in oxidation-

13、reduction reactionsMetals can be important for the structure of the enzymeMetal catalysisA Lewis acid is a species that can accept a pair of electrons for bondingZn functions as a Lewis acid in carbonic anhydraseMetals can act as Lewis acidsZn functions as a Lewis acid in carbonic anhydraseThis is t

14、he 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 catalyze the reaction. In addition to bond strain in the substrate, bond s

15、train 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 substrateCovalent catalysis accelerates reaction rates through the transient formation of a catalystsubstrate covalent bond. Usually,

16、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 form of catalysis is often also called nucleophilic catalysis.In addition, several coenzymes, notably thiamine pyrophosphate and pyridoxal phosphate, func

17、tion in association with their apoenzymes as covalent catalysts.Covalent catalysisIn covalent catalysis, the enzyme forms a transient covalent intermediate with the substrates of the reaction.Covalent catalysisBiologically important nucleophilic and electrophilic groupsBiologically important nucleop

18、hilic and electrophilic groups-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?Serine proteases - inactivated by diisopropyl fluorophosphateMetalloproteases - inactivated by EDTA or

19、o-phenanthroline (example - thermolysin)Carboxyl proteases - not active at neutral or slightly alkaline pH (example - HIV protease)Thiol proteases - inactivated by iodoacetamide or iodoacetateFour Major Classes of ProteasesGeneral Characteristics of ProteasesThey all go through a transition state in

20、 which the normally trigonal carbonyl carbon of the peptide bond es tetrahedral.This tetrahedral geometry occurs because of the temporary addition of a nucleophile.In the case of the serine and thiol proteases, the nucleophile is the serine hydroxyl or cysteine thiol at the active site whereas in th

21、e other two classes it is a water molecule.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. Best understood of the proteases All h

22、ave a peculiarly reactive Ser residueThere 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 One family is represented by the bacterial protease sub

23、tilisin, another by enzymes including chymotrypsin, trypsin, elastase, thrombin, kallikrein, etc. and, finally a family represented by wheat germ serine carboxypeptidase II Members in the trypsin family are involved in a range of cellular functions including blood clotting, complement activation, ho

24、rmone production, and fertilization Serine Proteasesall share an oxyanion holeall contain a catalytic triad - Serine, Histidine, and Aspartate: His directly abstracts a proton from Ser thereby converting it to an alkoxide ionThey differ most strikingly in their preference for amino acid side chains

25、on the C-side of Peptide bondTrypsin cleaves bonds only after Lys and Arg residues, chymotrypsin after large hydrophobic residues; the other proteases of this family have less distinct preferencesProteases can catalyze hydrolysis of both amides and estersGeneral Characteristics of Serine ProteasesThe serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the scissile