Ch 8 (pp. 141-144)

Enzyme Catalysis and Coenzymes

• Pepsin, a protease in the stomach, is a member of the aspartate protease superfamily, using two aspartate residues for acid-base catalysis.
• Aspartate proteases cannot use histidine for catalysis, unlike chymotrypsin.
• Enzymes in amino acid metabolism use pyridoxal phosphate derived from vitamin B6 for catalysis.
• Functional groups like coenzymes, metal ions, and metallocoenzymes are employed by enzymes for catalytic process.
• Almost all polar amino acids participate directly in catalysis, with some capable of covalent catalysis.
• Vitamin deficiencies result in loss of specific enzyme activities that depend on the coenzyme form of the vitamin.
• Coenzymes are tightly bound to enzymes and do not dissociate during the reaction.
• Ethanol inhibits the absorption of thiamin and displaces pyridoxal phosphate from its protein-binding sites.
• Coenzymes are usually synthesized from vitamins and participate in catalysis by providing functional groups.
• Coenzymes can be activation-transfer or oxidation-reduction coenzymes, with almost no catalytic power when not bound to the enzyme.
• Activation-transfer coenzymes participate in catalysis by forming a covalent bond with a portion of the substrate.
• Histidine's inability to extract a proton at low pH makes it unsuitable for general acid-base catalysis, unlike aspartic acid.

Enzyme Catalysis Mechanisms

• The serine hydroxyl group has a high pK, requiring enzyme structure to aid in proton removal and stabilization of the resulting oxygen anion at physiologic pH.
• The catalytic triad, consisting of aspartate, histidine, and serine, demonstrates cooperative interactions in the active site.
• The oxyanion tetrahedral transition-state complex in the reaction sequence is stabilized by hydrogen bonds with peptide backbone –NH groups.
• Enzymes like chymotrypsin form stable covalent intermediates during catalysis, involving serine or cysteine residues.
• The dissociating products of enzyme-catalyzed reactions are often destabilized by charge repulsion in the active site.
• Hydrolysis of the acyl–chymotrypsin intermediate involves the activation of water by the active-site histidine for a nucleophilic attack.
• Real enzymatic reactions result in a "multibump" energy diagram, with semistable covalent intermediates present as dips in the energy curve.
• Covalent catalysis involves the substrate being covalently linked to an amino acid side chain at the enzyme's active site during the reaction.
• Metal-ion catalysis, as seen in carbonic anhydrase, requires metal ions to allow catalysis to occur.
• Catalysis by approximation refers to the enzyme forcing substrates to bind in a manner that places reactive groups in the appropriate orientation for a reaction to occur.
• Nucleoside monophosphate kinases use catalysis by approximation to transfer a phosphate from a nucleoside triphosphate to a nucleoside monophosphate.
• Gastric acid in the stomach denatures proteins through disruption of ionic interactions and hydrogen bonding, lowering the pH to 1 to 2.

Chymotrypsin Catalytic Mechanism and Specificity

• Chymotrypsin is a serine protease that utilizes a serine in the active site to form a covalent intermediate during proteolysis.
• The hydrolysis reaction involves the addition of OH− from water to the carbonyl carbon of the peptide bond and an H+ to the N, cleaving the bond.
• Chymotrypsin catalyzes the reaction faster by using the hydroxyl group of a serine residue for the attack, activating it and stabilizing the oxyanion transition-state complexes.
• The reaction takes place in two stages: cleavage of the peptide bond in the substrate protein and hydrolysis of the acyl–enzyme intermediate.
• Specificity of binding to chymotrypsin involves hydrolyzing the peptide bond on the carbonyl side of specific amino acids in a denatured protein.
• The substrate-recognition site consists of a hydrophobic binding pocket that holds the hydrophobic amino acid contributing the carbonyl group of the scissile bond.
• Scissile-bond specificity is provided by the subsequent steps of the reaction, such as moving serine 195 into attacking position.
• In the first stage of the reaction, the peptide bond of the denatured protein substrate is cleaved as an active-site serine hydroxyl group attacks the carbonyl carbon.
• Aspartate and histidine cooperate in converting the hydroxyl group into a better nucleophilic attacking group by giving it a more negative charge.
• An active-site histidine acts as a base and abstracts a proton from the serine hydroxyl.
• The catalytic mechanism involves the formation of an oxyanion intermediate, stabilization of the N–H groups of serine 195 and glycine, and the use of histidine as a general base and acid catalyst.
• Chymotrypsin catalytic mechanism involves multiple catalytic strategies, including nucleophilic catalysis, acid–base catalysis, and stabilization of transition-state complexes.