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.
Tew
D G
DG
Departments of Molecular Recognition and Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, Essex, UK.
Boyd
H F
HF
Ashman
S
S
Theobald
C
C
Leach
C A
CA
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