Substrate Specificity in Short-Chain Phospholipid Analogs at the Active Site of Human Synovial Phospholipase A2

• Published in: Journal of medicinal chemistry 1994-11, Vol.37 (24), p.4118-4129
• Main Authors: Wheeler, T. N, Blanchard, Steven G, Fang, Frank, Gray-Nunez, Yolanda, Harris, Cole O, Lambert, Millard H, Mehrotra, Mukund M, Parks, Derek J, Andrew, R. C
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Models, Molecular; Phospholipases A - antagonists & inhibitors; Phospholipases A - metabolism; Phospholipases A2; Phospholipids - metabolism; Structure-Activity Relationship; Substrate Specificity; Synovial Fluid - enzymology
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm00050a009

The substrate specificity at the active site of recombinant human synovial fluid phospholipase A2 (hs-PLA2) was investigated by the preparation of a series of short-chain phospholipid analogs and measurement of their enzymatic hydrolysis at concentrations well below the critical micelle concentration. Substrates used in the study included 1,2-dihexanoylglycerophospholipids, 1,2-bis(alkanoylthio)glycerophospholipids, and 1-O-alkyl-2-(alkanoylthio)phospholipids. Turnover was observed for only a few of the 1,2-dihexanoylglycerophospholipids, and the rate of hydrolysis was very low, near the limit of detection of the assay. In contrast, selected 2-(alkanoylthio)-glycerophospholipids were hydrolyzed by hs-PLA2 at much higher rates at concentrations well below their critical micelle concentration (cmc). Thus, the 1,2-bis(hexanoylthio)glycerophosphatidylmethanol exhibits a k(cat)/K(M) = 1800 L mol-1 s-1. Over the calculated log P (cLogP) range of 3-9, cLogP and log(k(cat)/K(M) were linearly related for compounds with straight-chain sn-1 and sn-2 substituents. At comparable cLogP's, the sn-1 ethers and thioesters were hydrolyzed at comparable rates. A negative charge in the phosphate head group was required for enzyme activity. Unsaturation, aromaticity, and branching in the sn-2 substituent reduce turnover dramatically. The same structural modifications in the sn-1 substituent have less effect on turnover. Certain of these substrates, e.g., 1,2-bis(hexanoylthio)glycerophosphatidylmethanol, may be useful in assaying for active site inhibitors of PLA2. The structure--activity relationships established here for substrates should serve as a reference for the structure--activity relationships of substrate-based inhibitors.