The effect of a new penicillin analogue, 1-(2,6-dichlorophenyl)-4-methyl-5-pyrazolyl penicillin, designated pyrazocillin, on penicillinase (penicillin amido-β-lactamhydrolase, EC 126.96.36.199) from Bacillus cereus 569/H has been studied. Pyrazocillin inhibits the enzyme according to a "mixed" type inhibition. the Ki value was 1.1 · 10-2 M. The Km value as calculated from the hydrolysis velocity of pyrazocillin was higher than 1 · 10-2 M. In addition to the competitive inhibition the effect of the time-dependent inhibition of pyrazocillin has also been examined in detail. It has been established that penicillinase incubated with pyrazocillin but in the absence of substrate was partially inactivated. This inactivation can be reversed by dilution, but in contrast to the effect of penicillin analogues already known, the inactivating effect is not reversed by the substrate. Moreover, the conformation of the enzyme, which has low catalytic activity when partially inactivated by preincubation with pyrazocillin, is stabilized by the substrate, while with higher substrate concentrations the catalytic activity is further decreased. Based on this experimental evidence, the hypothesis of two substrate binding sites of the enzyme has been suggested.
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