Kinetics and Mechanism of Acyl Transfer by Penicillin G Acylase Comparing Acyl Donors Methyl Chloroacetate and Methyl Phenylacetate

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The enzymatic synthesis of 7-(chloroacetyl) amino-3-deacethylcephalosporanic acid (D-7-Cl-ACA) from 7-amino-3-deacethylcephalosporanic acud (D-7-ACA) and methyl chloroacetate (ClAAM) by penicillin G acylase (PA) (EC 3.5.1.11), which has the specific affinity for the phenylacetic acid moiety, was attempted and a 95% yield could be obtained using only 2.5 times the molar quantity of ClAAM. In contrast, the synthesis yield of 7-(phenylacetyl)-amino-3-deacetylcephalosporanic acid (D-7-Ph-ACA) from D7-ACA and methyl phenylacetate (PhAAM) by PA was 45% at a maximum. D-7-Cl-ACA and D-7-Ph-ACA did not differ markedly in their synthesis rates which were around 60 and 40 μmol/mg protein in the pH range of 7 to 9,respectively. Hydrolysis rates differed, however, and were around 2.6 and 44 μmol/mg protein in the pH range of 7 to 9,respectively. Both PhAA and ClAA they inhibited the synthesis noncompetitively; however, inhibited the hydrolysis competitively, and PhAA showed competitive inhibition of the hydrolysis of penicillin G(PenG). PhAA inhibited both synthesis and hydrolysis of D-7-Cl-ACA and D-R-Ph-ACA much more than ClAA; in particular, the K_i of PhAA for the D-7-Cl-ACA synthesis was 0.34 mM. In contrast, ClAA not only weakly inhibited the synthesis and hydrolysis of the chloroacetyl derivative of D-7-Cl-ACD, but only very weakly inhibited those of the phenylacetyl derivative of D-7-Ph-ACA. Differences in kinetic parameters are discussed using a mechanistic kinetic model for both synthesis and hydrolysis of D-7-Cl-ACA and D-7-Ph-ACA.
公益社団法人日本生物工学会の論文
1995-03-25

Kinetics and Mechanism of Acyl Transfer by Penicillin G Acylase Comparing Acyl Donors Methyl Chloroacetate and Methyl Phenylacetate

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