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 = 0.0
16 mM). The enzyme is partially inhibited at high benzylpenicillin con
centrations 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) th
an for the first one. Phenylmethylsulfonyl fluoride was shown to be a
very effective irreversible inhibitor, completely inactivating the pen
icillin acylase from A. faecalis in a few minutes at micromolar concen
trations; this compound was used for enzyme active site titration. The
absolute values of the determined kinetic parameters for enzymatic hy
drolysis of 6-nitro-3-phenylacetamidobenzoic acid (k(cat) = 95 s(-1) a
nd k(cat)/K-m = 2.1 x 10(-7) M-1 s(-1)) and benzylpenicillin (k(cat) =
54 s(-1) and k(cat)/K-m = 1.3 x 10(-7) M-1 s(-1)) by penicillin acyla
se from A. faecalis were shown to be highest of all the enzymes of thi
s family that have so far been studied. (C) 1997 Federation of Europea
n Biochemical Societies.