Kinetics and mechanism of inhibition of a serine beta-lactamase by O-aryloxycarbonyl hydroxamates

Abstract

The class C serine beta-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.

Figure 1

A. Loss of activity of the P99 β-lactamase (0.25 μM) as a function of time in the presence of 4 (0.7 μM). B. Titration of the activity of the P99 β-lactamase (1.0 μM) on addition of 4; v/v₀ is the fraction of enzyme activity remaining at a particular inhibitor/enzyme concentration ratio (I/E). C. Absorbance of the substrate cephalothin (0.2 mM) as a function of time on addition of the P99 β-lactamase (final concentration 1.9 nM) to a mixture of substrate and 4 (○, 0 μM;□, 9.5 μM;⋄, 28.7 μM;×, 47.9 μM). In all three graphs the points are experimental and the lines are derived from fitting the data to Schemes 1 and 2 (see text).

Figure 2

pH-rate profile for the hydrolysis of 1 (●) and 13 (○) in aqueous solution. The points are experimental and the lines are derived from fitting the data to Schemes 4a and 4b (see text).

Figure 3

pH-rate profile for the inactivation of the P99 μM β-lactamase by 1. The points are experimental and the line is derived from fitting the data to Schemes 5a or 5b (see text).

Figure 4

A. Fluorescence intensity change at 398 nm (excitation at 294 nm) on reaction of 3 (5.0 μM) with the P99 β-lactamase (1.0 μM). The fluorescence change reflects release of m-hydroxybenzoate. B. Fluorescence intensity change at 315 nm (excitation at 240 nm) on reaction of 8 (1.5 μM) with the P99 β-lactamase (0.5 μM). The fluorescence change reflects release of p-phenylbenzyl N-hydroxycarbamate. In both graphs the points are experimental and the line is derived from fitting the data to Scheme 1 (see text).

Figure 5

Absorbance changes with time on addition of the P99 β-lactamase (final concentration 1.0 μM) to mixtures of 1 (50 μM) and D-phenylalanine (▵, 0 mM;⋄, 5.0 mM;□, 10.0 mM;×, 20.0 mM;○, 40.0 mM). The points are experimental and the lines derived from fitting the data to Scheme 3 (see text).

Figure 6

Stereoviews of energy-minimized tetrahedral intermediate structures formed on reaction of the P99 β-lactamase with 1. A. During formation of the acyl-enzyme. B. During aminolysis of the acyl-enzyme by Lys 315, the crosslinking reaction. Only heavy atoms are shown.

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6