PH dependence of penicillin amidase enantioselectivity for charged substrates

Abstract

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the p K values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these p K values, a pH dependence of E is also expected. This was experimentally verified around these p K values. The S′ 1-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl- S/ R-Phe and for the racemic phenylacetyl- S, R-PhG. The S 1-stereospecificity was investigated for the hydrolysis of the enantiomeric S/ R-PhG-NH 2. The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5–9) was found to be a result of compensatory effects for binding and catalysis. The ratios k cat, S / k cat, R and K m, S / K m, R for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5–8. The dependence was stronger for the S′ 1- than for the S 1-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S′ 1-subsite than that of the substrate amino group in the S 1-subsite on the p K of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground–state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.