title: Substrate specificity in short-chain phospholipid analogs at the active site of human synovial phospholipase A2.
abstract: 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.
authors.affiliation: Division of Medicinal Chemistry, Glaxo Research Institute, Research Triangle Park, North Carolina 27709.
authors.forename: T N
authors.initials: TN
authors.lastname: Wheeler
authors.forename: S G
authors.initials: SG
authors.lastname: Blanchard
authors.forename: R C
authors.initials: RC
authors.lastname: Andrews
authors.forename: F
authors.initials: F
authors.lastname: Fang
authors.forename: Y
authors.initials: Y
authors.lastname: Gray-Nunez
authors.forename: C O
authors.initials: CO
authors.lastname: Harris
authors.forename: M H
authors.initials: MH
authors.lastname: Lambert
authors.forename: M M
authors.initials: MM
authors.lastname: Mehrotra
authors.forename: D J
authors.initials: DJ
authors.lastname: Parks
authors.forename: J A
authors.initials: JA
authors.lastname: Ray
mesh_terms: D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000201:enzymology*
chemical_list: D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2