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Molecular modeling was employed to explore the mechanism underlying the [alpha]-hydroxy group interaction with the class C [beta]-lactamase active site. The [alpha]-hydroxyalkyl moiety with ability to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates might find potential application when incorporated with novel inhibitors.

A series of new inhibitors is prepared to examine the kinetics and mechanisms of the inactivation reaction of serine [beta]-lactamase by O-aryloxycarbonyl hydroxamates. The studies have shown that the new series of analogues have reacted differently with the active site of a class C [beta]-lactamase than do normal substrates.

The study described various O-aryloxycarbonyl hydroxamates, which are used as the new [beta]-lactamase inhibitors that also lead to cross-linking of active site residues.

O-( I -Carboxy-l -alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both D- and L-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and D-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest. [ABSTRACT FROM AUTHOR]

The class C serine fi-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by O-aryloxycarbonyl hydroxamates. A series of these new inhibitors has been prepared to investigate the kinetics and mechanism of the inactivation reaction. A pH-rate profile for the reaction indicated that the reactive form of the inhibitor is neutral rather than anionic. The reaction rate is enhanced by electron- withdrawing aryloxy substituents and by hydrophobic substitution on both aryloxy and hydroxamate groups. Kinetics studies show that the rates of loss of the two possible leaving groups, aryloxide and hydroxamate, are essentially the same as the rate, of enzyme inactivation. Nucleophilic trapping experiments prove, however, that the aryl oxide is the first to leave. It is likely, therefore, that the rate-determining step of inactivation is the initial acylation reaction, most likely of the active site serine, yielding a hydroxamoyl- enzyme intermediate. This then partitions between hydrolysis and aminolysis by Lys 315, the latter to form an inactive, cross-linked active site. A previously described crystal structure of the inactivated enzyme shows a carbamate cross-link of Ser 64 and Lys 315. Structure-activity studies of the reported compounds suggest that they do not react at the enzyme active site in the same way as normal substrates. In particular, it appears that the initial acylation by these compounds does not involve the oxyanion hole, an unprecedented departure from known and presumed reactivity. Molecular modeling suggests that an alternative oxyanion hole may have been recruited, consisting of the side chain functional groups of Tyr 150 and Lys 315. Such an alternative mode of reaction may lead to the design of novel inhibitors. [ABSTRACT FROM AUTHOR]

The article describes a new type of β-lactamase inhibitor with an unusual mechanism of action involving the covalent cross-linking of active side residues. Results showed that inhibition of β-lactamases by cluvanic acid and penicillin sulfones has been shown to involve cross-linking of the active site serine to the conserved Ser130. Results of the study are presented and discussed.