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CMU BSC 03231 - lecture

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Biochemistry I Lecture 16 Oct 7, 20051Lecture 16: Enzyme Mechanism: Serine ProteasesAssigned reading in Campbell: Chapter 7.5-7.7Key Terms:• Transition State stabilization (enthalpic & entropic)• Regulation of Serine proteases: zymogens• Covalent Catalysis• Acyl-enzyme intermediate• Nucleophilic agent• Preferential binding of transition state complex• Catalytic triad (Ser, His, Asp)• Substrate specificity16.1 The transition state of the enzyme substrate complex is stabilized in two ways:1. Enthalpic - The enzyme transition-state complex is stabilized by direct interactions (e.g. H-bonds, electrostatic interactions) between the enzyme and the transition state. This reduces thefree energy of the transition state due to † DH (enthalpy).2. Entropic – Formation of the transition state requires a precise geometric arrangement ofsidechain groups. In the case of a reaction occurring in solution, this would require considerableordering of these chemical groups, i.e., a reduction in the entropy of the system, which isunfavorable.Biochemistry I Lecture 16 Oct 7, 20052In an enzyme, these groups are already in the correct position because of the way the proteinfolded, therefore there is no loss of entropy in the complex. For example, the serine proteasesutilize a serine, histidine, and aspartic acid to catalyze peptide bond hydrolysis. Consider thefollowing changes in entropy between the initial state and the transition state:16.2 Serine Proteases:Diverse role of Serine Proteases:Serine proteases play important roles in many processes, e.g. digestion of dietary protein, bloodclotting cascade, and in several pathways of differentiation and development. They are generallyproduced as inactive zymogens and auto-activate by peptide cleavage to form the active enzyme.Proteases active in digestion include: Trypsin, Chymotrypsin, Elastase.16.2 MechanismSerine proteases can hydrolyze either esters or peptide bonds utilizing mechanisms of covalentcatalysis and preferential binding of the transition state and by providing suitable reactivegroups near the peptide bond that is cleaved.Ester hydrolysis:Note that the brightyellow color of thep-nitrophenolate ionprovides a convenientway to monitor the rateof product formation. p-Nitrophenyl acetate p-Nitrophenolate (bright yellow)Peptide hydrolysis:(e.g. Chymotrypsin)16.3 Catalytic Mechanism: key features• Nucleophilic attack: a species that is electron rich (has a negative charge or anunshared electron pair) forms a bond with an electron deficient species. In the case ofthe peptide bond (or ester) the electronegativity of the oxygen makes the carbonyl carbonelectron deficient. OOONOCH3 H2ONOOO-CH3OO- H3N+OOOH3N+RO__ H2ONHH3N+OOOR-SerHisAspSSerHisAspSerHisAspSerHisAspSBiochemistry I Lecture 16 Oct 7, 20053Example of a nucleophilic attack that is also used by serine proteases:• Formation of an acyl-enzyme intermediate: Upon cleavage of the peptide bond, theacyl group of the substrate becomes covalently bound to the enzyme before it istransferred to a molecule of water.16.4 Substrate Specificity:Serine proteases utilize all of the intermolecular forces that we have discussed to bind theirsubstrates. In particular, the peptide bond to be cleaved often forms one or more hydrogenbonds with the enzyme. In addition to general recognition of the peptide, a particular serineprotease is specific for certain amino acids. The molecular nature of this specificity can beinferred from the structure of the active site:• Trypsin cleaves after Lys and Arg residues: Asp 189 in the active site of Trypsininteracts with the positive charge on Arg and Lys.• Chymotrypsin cleaves after aromatic (and large hydrophobic) residues: The activesite of the enzyme contains a hydrophobic pocket, formed in part by Met 192 ofChymotrypsin.• Elastase cleaves after small residues, especially glycine and alanine. Val and Thrresidues in the active site form a shallow binding pocket.Steps in catalytic cycle:1. Substrate binds2. Nucleophilic attack of the side chain oxygen of Ser 195 on the carbonyl carbon of thescissile bond (bond to be cleaved), forming a tetrahedral intermediate. Assistance fromHis 57 (proton transfer from Ser 195) and Asp 102. Tetrahedral intermediate or transitionstate is stabilized by amides of Ser 195 and Gly 193.3. Breakage of the peptide bond with assistance from His 57 (proton transfer to the newamino terminus). Release of the first product.4. Acyl-intermediate: Note that the substrate is covalently attached to the active siteSer 195.5. Nucleophilic attack of water on the acyl-enzyme intermediate with assistance of His 57and formation of the tetrahedral intermediate.Decomposition of acyl-intermediate and release of the second product. Enzyme is in the sameform as at the start!Biochemistry I Lecture 16 Oct 7, 20054Mechanism of Serine Proteases: ONHR1NOR2 NOR2ONHR1OHHH NNHSerOHOO_Substrate1NHR2R1OHHNN NNHOO_NHR2R1OHHNNSerOH_2 NNHOO_NHR2R1OHHNNSerOH1st Product3 NNHOO_HOHR1OHHNNSerO4 OO_R1OSerOOH_HHNN5NNHH OO_HHNN6NNHHSerOHOR1O2nd


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