Kinetic study of penicillin acylase from Alcaligenes faecalis
1997; Wiley; Volume: 417; Issue: 3 Linguagem: Inglês
10.1016/s0014-5793(97)01289-1
ISSN1873-3468
AutoresVytas K. Švedas, Д. Т. Гуранда, Luuk M. van Langen, Fred van Rantwijk, R. Ann Sheldon,
Tópico(s)Biochemical and Molecular Research
ResumoPenicillin 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.
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