Substrate chirality and specificity of diacylglycerol kinases and the multisubstrate lipid kinase.

Richard M Epand, Yulia V Shulga, Heath C Timmons, Alexandra L Perri, Jitendra D Belani, Kirishanth Perinpanathan, Laura Beth Johnson-McIntire, Sandra Bajjalieh, Armela O Dicu, Cynthia Elias, Scott D Rychnovsky, Matthew K Topham

Index: Biochemistry 46(49) , 14225-31, (2007)

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Abstract

The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times more rapidly than 2,3-dioleoylglycerol. The alpha and zeta isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective epsilon isoform. The inhibition by 2,3-dioleoylglycerol is uncompetitive. This corresponds to a kinetic scheme in which the inhibitor can bind to the enzyme-substrate complex, but not to the free enzyme. Our data indicate that despite their similar structures, 1,2-dioleoylglycerol and 2,3-dioleoylglycerol do not compete for the active site of these three isoforms of diacylglycerol kinase. We suggest that the 2,3-dioleoylglycerol binds to a site on the alpha and zeta isoforms of diacylglycerol kinase that is exposed as a consequence of the substrate binding to the active site. The chiral specificity of these enzymes thus mimics the substrate specificity, with MuLK being the least selective and the epsilon isoform of diacylglycerol kinase exhibiting the greatest selectivity.