MECHANISM OF INHIBITION OF LDL PHOSPHOLIPASE A(2) BY MONOCYCLIC-BETA-LACTAMS - BURST KINETICS AND THE EFFECT OF STEREOCHEMISTRY

Investigation of the inhibition of LDL-associated phospholipase A(2) b y monocyclic beta-lactams has shown that LDL phospholipase A(2) is cap able of hydrolyzing monocyclic-beta-lactams by a mechanism which share s many similarities to the hydrolysis of beta-lactams by beta-lactamas es, We believe that this is the first demonstration of a serine-depend ent lipase being able to hydrolyze an amide bond. Although 4-(phenylth io)-N-(4-phenyl-2-oxobutyl)az SB-216477, and its enantiomers are relat ively modest covalent inactivators with k(obs)/[I] = 46 M-1 s(-1) for the R enantiomer, analysis of the kinetics of inactivation and reactiv ation shows that these compounds act as slow-turnover substrates, pres umably via an acylation-deacylation mechanism. The detection of a supr astoichiometric 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 SE-216477 shows that LDL-associated phospholipase A(2) is sensitive to the p-lactam s tereochemistry 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.