Mechanism of Inhibition of LDL Phospholipase A2 by Monocyclic-β-lactams. Burst Kinetics and the Effect of Stereochemistry

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Abstract
Investigation of the inhibition of LDL-associated phospholipase A2 by monocyclic β-lactams has shown that LDL phospholipase A2 is capable of hydrolyzing monocyclic-β-lactams by a mechanism which shares many similarities to the hydrolysis of β-lactams by β-lactamases. We believe that this is the first demonstration of a serine-dependent lipase being able to hydrolyze an amide bond. Although 4-(phenylthio)-N-(4-phenyl-2-oxobutyl)azetidin-2-one, SB-216477, and its enantiomers are relatively modest covalent inactivators with kobs/[I] = 46 M-1 s-1 for the R enantiomer, analysis of the kinetics of inactivation and reactivation shows that these compounds act as slow-turnover substrates, presumably via an acylation−deacylation mechanism. The detection of a suprastoichiometric burst indicates that the pathway must be branched with the branching giving rise to the slow reactivation via a more stable covalent intermediate. Study of the two enantiomers of SB-216477 shows that LDL-associated phospholipase A2 is sensitive to the β-lactam stereochemistry at C4. However, a common achiral intermediate is formed along the turnover pathway, and this must be at or immediately prior to the branch point.