title: Kinetic study of penicillin acylase from Alcaligenes faecalis.
abstract: 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.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia. vytas@enzyme.genebee.msu.su
authors.forename: V
authors.initials: V
authors.lastname: Svedas
authors.forename: D
authors.initials: D
authors.lastname: Guranda
authors.forename: L
authors.initials: L
authors.lastname: van Langen
authors.forename: F
authors.initials: F
authors.lastname: van Rantwijk
authors.forename: R
authors.initials: R
authors.lastname: Sheldon
mesh_terms: D000421:Alcaligenes / Q000201:enzymology*; D003124:Colorimetry / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010399:Penicillin Amidase / Q000378:metabolism*; D010400:Penicillin G / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D010399:Penicillin Amidase; D010400:Penicillin G