Part Four: Examples of Enzymatic Reactions.

• The chymotrypsin mechanism involves acylation and deacylation of a Ser residue.
• A protease is an enzyme that catalyzes the hydrolytic cleavage of peptide bonds, such as bovine pancreatic chymotrypsin.
• The acylation phase of the chymotrypsin mechanism is where the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme.
• The deacylation phase of the chymotrypsin mechanism is the ester linkage is hydrolyzed and the nonacylated enzyme is regenerated.
• In the chymotrypsin mechanism, the presence of a burst suggested that turnover of the enzyme was limited by subsequent, slower deacylation step.
• The optimal activity for chymotrypsin catalyzed reactions is pH 8, where His is unprotonated and Ile is protonated. The transition just above pH 7 is due to changes of k_cat from the protonation of His. The transition above pH 8.5 is due to changes in 1 / K_m from the ionization of the alpha amino group in Ile.
• Serine proteases are proteases with a Ser residue that acts as a nucelophile.
• The acylation phase nucleophile of the chymotrypsin serine protease is the oxygen of Ser.
• The catalytic triad is a hydrogen bonding network. In chymotrypsin, Ser is linked to His and Asp.
• The first step of the chymotrypsin mechanism is when the substrate binds, the side chain adjacent to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack.
• The second step of the chymotrypsin mechanism is the interaction of Ser and His generates a strongly nucleophilic alkoxide ion on Ser, the ion attacks the peptide carbonyl group, forming a tetrahedral acyl enzyme, and this is accompanied by the formation of a short lived negative charge on the carbonyl oxygen of the substrate, which is stabilized by hydrogen bonding in the oxyanion hole.
• The third step of the chymotrypsin is the instability of the negative charge on the substrate carbonyl oxygen leads to collapse of the tetrahedral intermediate. Reformation of a double bond with carbon displaces the bond between carbon and the amino acid group of the peptide linkage, breaking the peptide bond. The amino leaving group is protonated by His, facilitating its displacement.
• The fourth step of the chymotrypsin mechanism is an incoming water molecule is deprotonated by general base catalysis, generating a strongly nucleophilic hydroxide ion. Attack of hydroxide on the ester linkage of the acyl enzyme generates a second tetrahedral intermediate, with oxygen in the oxyanion hole again taking on a negative charge.
• In the fifth step of the chymotrypsin mechanism, there is the collapse of the tetrahedral intermediate that forms the second product, a carboxylate anion, and displaces Ser.
• The sixth step of the chymotrypsin mechanism is the dissociation of the second product from the active site regenerates free enzyme.
• The mechanism of action of an HIV protease begins by, with the aid of general base catalysis, water attacks the carbonyl carbon, generating a tetrahedral intermediate stabilized by hydrogen bonding. Then the tetrahedral intermediate collapses and the amino acid leaving group is protonated as it is expellled.
• HIV protease inhibitors form noncovalent complexes with the enzyme. This works as transition state analogs, and thus is irreversible inhibition.
• Hexokinase undergoes induced fit on substrate binding.
• Yeast hexokinase is a bisubstrate enzyme that catalyzes the reversible reaction.
• When glucose and Mg ATP bind, the binding energy derived induces a conformation change in hexokinase to the catalytically active form.
• Xylose cannot be phosphorylated, so its binding induces a change in hexokinase to its active conformation.
• The active site amino residues of hexokinase participates in general acid base catalysis and transition state stabilization
• Enolase catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate.
• The mechanism of the enolase reaction begins when Lys abstracts a proton by general base catalysis. Two Mg ions stabilize the resulting enolate intermediate. Then, Glu facilitates elimination of the hydroxyl group by general acid catalysis.
• Peptidoglycan is a major component of bacterial cell wall. It consists of polysaccharides and peptides cross linked in several steps that include a transpeptidase reaction.
• Beta-lactam antibiotics tend to bind to the active site of transpeptidase. The covalent complex irreversible inactivates the enzyme, blocking synthesis of the cell wall.
• Beta-lactamases are enzymes the cleave beta-lactam antibiotics.
• Bacteria that express beta-lactamases become resistant to beta-lactam antibiotics.
• Compounds that mimic the structure of a beta-lactam antibiotic can inactivate beta-lactamases.