Kinetic study of penicillin acylase from Alcaligenes faecalis

Abstract

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, K m=0.0042 mM) and colorimetric (6-nitro-3-phenylacetamidobenzoic acid, K m=0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid ( K i=0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower ( K S′=54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6-nitro-3-phenylacetamidobenzoic acid ( k cat=95 s −1 and k cat/ K m=2.1×10 −7 M −1 s −1) and benzylpenicillin ( k cat=54 s −1 and k cat/ K m=1.3×10 −7 M −1 s −1) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.