Mechanism of inhibition of LDL phospholipase A2 by monocyclic-beta-lactams. Burst kinetics and the effect of stereochemistry.
Investigation of the inhibition of LDL-associated phospholipase A2 by monocyclic beta-lactams has shown that LDL phospholipase A2 is capable of hydrolyzing monocyclic-beta-lactams by a mechanism which shares many similarities to the hydrolysis of beta-lactams by beta-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 beta-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.
Departments of Molecular Recognition and Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, Essex, UK.
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Tew
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Boyd
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Ashman
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Theobald
C A
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Leach
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D001384:Azetidines / Q000378:metabolism / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D007769:Lactams; D008077:Lipoproteins, LDL / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054467:Phospholipases A2; D013057:Spectrum Analysis; D013237:Stereoisomerism; D013379:Substrate Specificity; D047090:beta-Lactams / Q000378:metabolism / Q000494:pharmacology*
D001384:Azetidines; D007769:Lactams; D008077:Lipoproteins, LDL; C113572:SB-216477; D047090:beta-Lactams; D010741:Phospholipases A; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase