Mechanism of inactivation of TEM-1 β-lactamase by 6-acetylmethylenepenicillanic acid

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Abstract
The interaction between 6-acetylmethylenepenicillanic acid (compound Ro 15-1903; AMPA) and TEM-1 beta-lactamase was investigated in order to elucidate the mechanism of action of AMPA. Formation of the enzyme-inhibitor complex (EA) was accompanied by a shift of the absorbance maximum from 292 nm to 303 nm and an increase in the absorption. Regeneration of activity was very slow and incomplete, reaching about one-third of the initial activity after 48 h at 37 degrees C. This behaviour indicated a branched pathway of the decay of the inactivated enzyme. Kinetic and isoelectric-focusing experiments proved this assumption. The first-order constant of regeneration of active enzyme was 6 × 10(-6)-10 × 10(-6) s-1, whereas the rate constant leading to inactive enzyme (EA') was 10 × 10(-6)-15 × 10(-6) s-1 at pH 7.0. Both constants became larger at higher pH. Inactive enzyme (EA') consisted of two major species, with pI 5.36 (EA'1) and 5.30 (EA'2). The former increased at the beginning of incubation but decreased after prolonged incubation. From consideration of these results and previous data [Arisawa & Then (1983) Biochem. J. 209, 609-615], a likely mechanism of inactivation of TEM-1 beta-lactamase by AMPA is discussed.