Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, Km = 0.0042 mM) and colorimetric (6-nitro-3-phenylacetamidobenzoic acid, Km = 0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (Ki = 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)/Km = 2.1 x 10(-7) M(-1) s(-1)) and benzylpenicillin (k(cat) = 54 s(-1) and k(cat)/Km = 1.3 x 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.