Substrate specificity in short-chain phospholipid analogs at the active site of human synovial phospholipase A2

Article abstract of 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.