Inhibition Effects in the Hydrolysis Reactions of Esters and Peptides Catalyzed by Carboxypeptidase A: An Example of Cooperative Binding Effects with a Monomeric Enzyme
✍ Scribed by Abdulkader Rahmo; Thomas H. Fife
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 151 KB
- Volume
- 28
- Category
- Article
- ISSN
- 0045-2068
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✦ Synopsis
N-benzoyl-L-phenylalanyl-L-phenylalanine is an excellent peptide substrate for carboxypeptidase A; at 30°C and pH 7.5, K m is 2.6 × 10 -5 M while k cat is 177 s -1 (k cat /K m = 6.8 × 10 6 M -1 s -1 ). Indole-3-acetic acid is a noncompetitive or mixed inhibitor towards the peptide and toward hippuryl-L-phenylalanine; plots of E/V vs [Inhibitor] are linear. N-Benzoyl-L-phenylalanine is a competitive inhibitor of peptide hydrolysis, and plots of E/V vs [Inhibitor] are again linear. One molecule of inhibitor binds per active site, and these inhibitors bind in different sites. At constant peptide substrate concentration and a series of constant concentrations of indole-3-acetic acid, plots of E/V vs the concentration of Nbenzoyl-L-phenylalanine are linear and intersect behind the E/V axis and above the [Inhibitor] axis. This shows that both inhibitors can bind simultaneously and that binding of one facilitates the binding of the other (β = 0.18). Employing the ester substrate hippuryl-DL,βphenyllactate, the same type of behavior is observed in the reverse sense; N-benzoyl-Lphenylalanine is a linear noncompetitive inhibitor and indole-3-acetic acid is a linear competitive inhibitor. Again the two inhibitor plot is linear and intersects above the [Inhibitor] axis (β = 0.12). Previous X-ray crystallographic studies have indicated that indole-3-acetic acid binds in the hydrophobic pocket of the S 1 ′ site, while N-benzoyl-L-phenylalanine binds in the S 1 -S 2 site. The product complex for hydrolysis of N-benzoyl-L-phenylalanyl-L-phenylalanine (phenylalanine + N-benzoyl-L-phenylalanine) occupies both of these sites. However, the present work shows that the peptide substrate does not bind to the enzyme at pH 7.5 so as to be competitive with indole-3-acetic acid. The binding sites may be formed via conformational changes induced or stabilized by substrate and product binding.