title: 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 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 of E is also expected. This was experimentally verified around these pK 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 pK 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.
authors.affiliation: Department of Biotechnology II, Technical University of Hamburg-Harburg, Denickestr. 15, D-21071, Hamburg, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Lummer
authors.forename: A
authors.initials: A
authors.lastname: Rieks
authors.forename: B
authors.initials: B
authors.lastname: Galunsky
authors.forename: V
authors.initials: V
authors.lastname: Kasche
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010399:Penicillin Amidase / Q000737:chemistry*; D011786:Quality Control; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010399:Penicillin Amidase