SUBSTRATE-SPECIFICITY IN SHORT-CHAIN PHOSPHOLIPID ANALOGS AT THE ACTIVE-SITE OF HUMAN SYNOVIAL PHOSPHOLIPASE A(2)

The substrate specificity at the active site of recombinant human syno vial fluid phospholipase A(2) (hs-PLA(2)) was investigated by the prep aration of a series of short-chain phospholipid analogs and measuremen t of their enzymatic hydrolysis at concentrations well below the criti cal micelle concentration. Substrates used in the study included 1,2-d ihexanoylglycerophospholipids, 1,2-bis(alkanoylthio)glycerophospholipi ds, and 1-O-alkyl-2-(alkanoylthio)phospholipids. Turnover was observed for only a few of the 1,2-dihexanoylglycerophospholipids, and the rat e of hydrolysis was very low, near the limit of detection of the assay . In contrast, selected 2-(alkanoylthio)glycerophospholipids were hydr olyzed by hs-PLA(2) at much higher rates at concentrations well below their critical micelle concentration (cmc). Thus, the 1,2-bis(hexanoyl thio)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 thi oesters were hydrolyzed at comparable rates. A negative charge in the phosphate head group was required for enzyme activity. Unsaturation, a romaticity, and branching in the sn-2 substituent reduce turnover dram atically. The same structural modifications in the sn-1 substituent ha ve less effect on turnover. Certain of these substrates, e.g., 1,2-bis (hexanoylthio)glycerophosphatidylmethanol, may be useful in assaying f or active site inhibitors of PLA(2). The structure-activity relationsh ips established here for substrates should serve as a reference for th e structure-activity relationships of substrate-based inhibitors.