Kinetics of Action of a Two-Stage Pro-Inhibitor of Serine β-Lactamases

β-Lactamase inhibitors are important in medicine in the protection of β-lactam antibiotics from β-lactamase-catalyzed destruction. The most effective inhibitors of serine β-lactamases covalently modify the enzyme active site. We have recently studied O-acyl and O-phosphyl hydroxamates as a new class of such inhibitors. In this paper, we describe our studies of the N-acyl derivatives of a cyclic O-acyl hydroxamic acid, 3H-benzo[d][1,2]oxazine-1,4-dione, and, in particular, the N-tert-butoxycarbonyl derivative. This compound is not a β-lactamase inhibitor itself but undergoes spontaneous hydrolysis in aqueous solution, yielding an O-phthaloyl hydroxamic acid, which is a β-lactamase inhibitor. This compound spontaneously, but reversibly, cyclizes in solution to form phthalic anhydride, which is also a β-lactamase inhibitor. Both inhibitors react to form the same transiently stable phthaloyl–enzyme complex. Thus, we have a two-step cascade, beginning with a pro-inhibitor, in which each step leads to a differe...