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Kinetic study of penicillin acylase from Alcaligenes faecalis
Author(s) -
Švedas Vytas,
Guranda Dorel,
van Langen Luuk,
van Rantwijk Fred,
Sheldon Roger
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01289-1
Subject(s) - alcaligenes faecalis , benzylpenicillin , chemistry , penicillin , penicillin amidase , substrate (aquarium) , phenylacetic acid , hydrolysis , titration , enzyme , nuclear chemistry , chromatography , stereochemistry , biochemistry , bacteria , organic chemistry , antibiotics , biology , ecology , genetics
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.