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Izumenolide (I), which was isolated from Micrornonospora chalcea subsp. izumensis,1) has recently been reported to be a specific inhibitor of i3-lactamase, especially those enzymes from Gram-negative bacteria 2). In studies with TEM-2 i3-lactamase from Escherichia coli, incubation of izumenolide and enzyme at molar ratios greater than ten resulted in a complete, and irreversible, loss of activity. This specificity is greater than that observed with clavulanic acid which requires about 115 moles of inhibitor per mole of enzyme before the TEM-2 /3-lactamase is inactivated3). In contrast to other naturally-occurring j3lactamase inhibitors such as clavulanic acid) and the olivanic acids5 -8), izumenolide is not a i3lactam, but a macrolide containing sulfate ester groups9). Because the specificity of the molecule appears to be associated with the sulfate ester functionality2), other enzyme inhibitors containing sulfate groups were studied. Panosialin (II), which is widely distributed among Streptomyces strains (WELLS and SYKES, unpublished observations), is a mixture of compounds reported to inhibit sialidase, acid phosphatase and polygalacturonase10). SDS (sodium dodecyl sulfate, III) has long been recognized both as a general enzyme inhibitor11) and as an inhibitor of ,3lactamases12). Both II and III are active presumably because of their detergent-like properties. In order to determine whether izumenolide resembles SDS and panosialin, the mode of action of these compounds was studied with respect to their inhibition of TEM-2 13-lactamase. TEM-2 jI-lactamase was purified to homogeneity using a modification of the method described by MELLING and SCOTT13). Experimental procedures, including details regarding the spectrophotometric assay of ,3-lactamase activity, are described elsewhere 2). SDS was obtained from Sigma, panosialin and izumenolide were isolated from our screening program. As seen in Table 1, SDS and panosialin behave as pure competitive inhibitors of TEM-2 I,lactamase, indicating that these anionic detergents bind at the ; -lactamase active site. However, initial reaction rates in the presence of izumenolide exhibited either mixed kinetic behavior or hyperbolic competitive kinetics2), suggesting that the binding site for izumenolide may extend beyond the enzymatic active site. Unlike izumenolide and other 13-lactamase inactivators3) no evidence was observed with either SDS or panosialin for progressive inhibition of the enzyme. Thus, the inhibitory characteristics of the two detergent molecules are significantly different from those of izumenolide. Multiple inhibition studies were conducted using carbenicillin (a poor substrate) and either SDS or izumenolide, according to the procedure described by YONETANI and THEORELL14). As seen in Fig. I parallel plots for control enzyme and SDS-treated enzyme indicate that carbenicillin and SDS bind at the same site, whereas the intersecting lines for control enzyme and izumenolide-treated enzyme show that nonidentical binding sites are involved for izumenolide and carbenicillin. These results are consistent with the kinetic studies, which show that SDS binds at the catalytic center whereas izumenolide binding involves another site on the enzyme. Together, these studies indicate that the TEM2 /3-lactamase recognizes sulfate-containing molecules such as SDS and panosialin at the catalytic (1)

Inhibition of Escherichia coli tem-2-.BETA.-lactamase by the sulfated compounds izumenolide, panosialin and sodium dodecyl sulfate.