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Unformatted text preview: Chapter 9: Stryer Lecture 9 Catalytic strategies What are the sources of catalytic power and speciﬁcity? What are the roles of the protein side chains? From Ch. 8: binding energy – the free energy released in the formation of a large number of weak interactions between the enzyme and its substrate. It serves two purposes: 1) establish substrate speciﬁcity, 2) increase catalytic efﬁciency. Interactions between the enzyme and substrate in its transition state are most important. Why? Lower the activation energy. Induced ﬁt – changes in the enzyme and substrate to facilitate catalysis. What are the types of strategies that enzymes use? • covalent catalysis • general acid-base catalysis • catalysis by approximation • metal-ion catalysis Overview: Catalytic strategies & mechanisms: • serine proteases • carbonic anhydrase • restriction enzymes • NMP kinases Acid-base catalysis General acid catalysis - a process in which partial or complete proton transfer from a Brønsted acid lowers the free energy of a reactions transition state. General base catalysis - a process in which partial or complete proton abstraction by a Brønsted acid lowers the free energy of a reactions transition state. Covalent catalysis Covalent catalysis - rate acceleration achieved through the transient formation of a covalent enzyme-substrate complex Good side chains for covalent catalysis must combine high nucleophilicity and the ability to act as a good leaving group Groups that are known to act in covalent catalysis -His, Cys, Asp, Glu, Ser and Lys -also some cofactors (pyridoxal phosphate, thiamine pyrophosphate, biotin, etc.) Example - Serine Proteases - acyl-enzyme intermediate Compare this mechanism with an acid-catalyzed cleavage. Metal-ion catalysis Nearly 1/3 of all enzymes require a metal ion cofactor for activity!! Common roles of metal-ion cofactors:
• Bind and orient substrates • Mediate oxidation-reduction reactions (1 or 2 e- depending on ion) • Stabilize anionic intermediates (charge shielding) • Activate water to make it a better nucleophile
His Zn His
2+ Catalysis by approximation Many reactions include two distinct substrates. In these cases, the reaction rate may be considerably enhanced by bringing the two substrate together along a single binding surface on an enzyme. Example: NMP kinase - brings two nucleotides together to facilitate phosphoryl transfer O CO2H CH3C
substrate OHis H C CH3C O CH3C C O + H2O -H+
H O His H His His Zn
2+ CH3C CO2H need 3 ! 105 M acetic acid to get same effective rate constant as the intramolecular reaction with succinic acid; pure acetic acid is only 17.5 M Proximity and orientation effects Effective concentration - preorganization of the active site makes collisions between two substrates more common and more likely to be productive (i.e. yield product)
Enzymes convert second order reactions into ﬁrst order reactions A+B A B k1 k2 AB A B k1 units = M-1 s-1 k2 units = s- 1 Catalytic functions of common ionizable amino acids Amino acid Asp Glu His Cys Tyr Lys Arg Ser Reactive Group COOCOOimidazole -CH2SH Ph-OH R-NH3
+ Approx. p Ka 4-5 4-5 6-7 8-9.5 9.5 - 10 !10 ! 12 !16 Charge pH 7 -1 -1 0 0 0 +1 +1 0 Common function cation binding/H+ transfer cation binding/H+ transfer H+ transfer covalent binding acyl grps H-bonding anion binding/H+ transfer anion binding covalent binding acyl grps guanidinium -CH2OH Effective concentration (units = M) = kcat/kuncat = k2/k1 • Proximity worth approximately factor of 5 • Fixed orientation worth factor of ! 100 • Immobilization of translational /rotational motions factors of >105 Enzyme mechanism Enzyme activity depends on pH pH proﬁles why?? more important concepts to remember Proteases Chymotrypsin: an example of covalent catalysis Chymotrypsin cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids. What nucleophile is used to attack the unreactive carbon? very slow: half-life at pH 7 is ~10-1000 years! Remember from Ch. 2: proteins are difﬁcult to hydrolyze; proteins that have served their purposes are degraded so that their constituent amino acids can be recycled or used in various metabolic pathways What is the signiﬁcance of the resonance structure with respect to reactivity of the peptide bond? Chymotrypsin X-ray structure: three chains and a catalytic triad Enzyme mechanism Example: protease chymotrypsin The catalytic triad (Asp 102, His 57, Ser 195) converts serine 195 into a potent nucleophile !- catalytic triad Hint about the mechanism: chymotrypsin is inactivated by treatment with diisopropylﬂuoridate (DIPF), which reacts with serine 195 (out of 28 possible serine residues); called mechanismbased inactivators Another hint about the mechanism: N-acetyl-L-phenylalanine pnitrophenyl ester is hydrolyzed by chymotrypsin to give a yellow product, p-nitrophenolate at pH 7.0. This reaction can be monitored to provide information about the enzyme kinetics. (nerve gas) Chymotrypsin: an example of covalent catalysis Hydrolysis by chymotrypsin takes place in two stages: 1) acylation to form a covalent acyl-enzyme intermediate, 2) deacylation to regenerate free enzyme Chymotrypsin mechanism Active site pocket deﬁnes substrate speciﬁcity Chymotrypsin mechanism Chymotrypsin mechanism important points: • His is acting as a general base • oxyanion hole (tetrahedral intermediate) is stabilized by interactions with the enzyme Chymotrypsin mechanism Chymotrypsin mechanism release of the amine component • collapse to acyl-intermediate • His is acting as a general acid Chymotrypsin mechanism Chymotrypsin mechanism • role of water in deacylation • His is acting as a general base Chymotrypsin mechanism Chymotrypsin mechanism release of carboxylic acid component The enzyme is ready for another round • collapse of tetrahedral intermediate • His is acting as a general acid Chymotrypsin mechanism Chymotrypsin speciﬁcity The sites of substrate interaction are noted as follows (the scissile bond is in red): Structural similarity of chymotrypsin & trypsin Catalytic triads are found in other enzymes: Catalytic triad and oxyanion hole of subtilisin Carboxypeptidase II How can you test the role of the individual amino acids in the catalytic triad (this case is subtilisin)? site-directed mutagenesis *note the Y axis is a log scale HIV protease: a dimeric aspartyl protease • note two Asp residues (one in each half of the homodimer) • involved in general acid-base catalysis compare the peptide substrate to the HIV protease inhibitor, Indinavir Representative aspartic proteases Speciﬁc interactions involved in substrate speciﬁcity
HIV PR Consenesus Recognition Site: (S/T)XX(F/Y)*P(I/V) Porcine pepsin HIV-1 Protease Note: conserved water use of glycines in backbone recognition of substrate many interactions made by protease backbone Meek (1998) in Comprehensive Biological Catalysis, Sinnott Ed. Ch. 8 Meek (1998) in Comprehensive Biological Catalysis, Sinnott Ed. Ch. 8 Intermediates on the drug discovery pathway to Saquinavir Substrate speciﬁcity determinants of HIV-PR
HIV PR Consenesus Recognition Site: (S/T)XX(F/Y)*P(I/V) Wlodawer and Vondarsek (1998) Annu. Rev. Biophys. Biomol. Struct. 27: 249-84 Wlodawer and Vondarsek (1998) Annu. Rev. Biophys. Biomol. Struct. 27: 249-84 FDA approved HIV-PR inhibitors (1998)
HIV PR Consensus Recognition Site: (S/T)XX(F/Y)*P(I/V) HIV protease complexed with Indinavir competitive inhibition transition state mimic Wlodawer and Vondarsek (1998) Annu. Rev. Biophys. Biomol. Struct. 27: 249-84 • one Asp has a relatively low pKa, while the other has a relatively high pKa • deprotonated Asp is a general base, accepting water proton, whereas the other Asp is a general acid, facilitating formation of the tetrahedral intermediate Carbonic anhydrase Ch. 7 106 s-1 Effect of pH on carbonic anhydrase activity consider the pKa values of active site amino acid residues; in this case the metal ion pKa is also important Note the zinc ion bound to the imidazole rings of three His residues Mechanism of carbonic anhydrase water deprotonation carbon dioxide binding displacement of bicarbonate by water Synthetic analogue of carbonic anhydrase nucleophilic attack of hydroxide on carbon dioxide This synthetic model for carbonic anhydrase is capable of binding zinc. The zinc complex of this ligand accelerates the hydration of carbon dioxide more than 100-fold under certain conditions. The presumed active complex binds zinc and one water molecule. Restriction enzymes: highly speciﬁc DNA cleaving agents cleavage occurs by in-line displacement of the 3’-oxygen by a magnesium-activated water pentacoordinate transition state The recognition sequence for the restriction enzyme EcoRV and sites of base methylation, which prevents cleavage. Stereochemistry of the cleaved DNA: use of DNA analogs Role of the metal ion Are there other mechanistic possibilities? Role of the metal ion Methylation of adenine disrupts hydrogen bonds with the enzyme and prevents DNA hydrolysis Nucleoside monophosphate kinase: phosphoryl transfer NMP kinase structure: role of the P loop NMP kinase structure: role of the P loop The core domain with P loop in green: the P loop interacts with phosphoryl of ATP. Note conserved Lys. ATP-Mg2+ complex ATP induces large conformational changes in adenylate kinase P-Loop NTPase domains are present in a range of important proteins ...
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- Fall '08