pH dependence of penicillin amidase enantioselectivity for charged substrates

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantit ative measure for enzyme stereospecificity) was studied for penicillin amid ase catalysed hydrolysis of charged enantiomeric substrates. Theoretical an alysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence o f E is also expected. This was experimentally verified around these pK valu es. The S'(1)-stereospecificity of penicillin amidase was studied for the h ydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phen ylacetyl-S,R-PhG. The S-1-stereospecificity was investigated for the hydrol ysis of the enantiomeric SIR-PhG-NH2. 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,K-S/K-m,K-R for the hydrolysis of the enantiomeri c 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 influ ence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S-1-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was us ed to discuss the importance of ground-state interactions for discriminatio n between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better bindin g must not be fundamentally inhibitory. (C) 1999 Elsevier Science B.V. All rights reserved.