Ostroski Michele, Tu-Sekine Becky, Raben Daniel M
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205-2185, USA.
Biochemistry. 2005 Aug 2;44(30):10199-207. doi: 10.1021/bi0507276.
Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-theta, DGKA, was identified in Dictyostelium discoideum. It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis-Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).
二酰基甘油激酶（DGKs）催化二酰基甘油的ATP依赖性磷酸化反应以生成磷脂酸，并且已经在原核生物和真核生物中进行了研究。最近，在盘基网柄菌中鉴定出一种与人类DGK-θ、DGKA显著相似的蛋白质。当使用中链二酰基甘油1,2-二辛酰基-sn-甘油（DiC8）进行检测时，它已被证明具有DGK活性。要全面了解DGK的催化和调节机制以及生理作用，需要了解其生化和动力学特性。本报告介绍了对DGKA这些特性的分析。该酶以米氏动力学方式催化DiC8和另一种中链二酰基甘油DiC6（1,2-二己酰基-sn-甘油）的磷酸化反应。有趣的是，DGKA使用生理相关长链二酰基甘油的动力学取决于底物表面浓度和所使用的去污剂。当表面底物浓度等于或低于3.5 mol%时，DGKA在辛基葡糖苷混合胶束中对总体底物浓度（1,2-二油酰基-sn-甘油）表现出米氏动力学。然而，在较高的表面浓度下，初始速度与总体底物浓度之间呈S形关系。相比之下，在Triton X-100混合胶束中，DGKA在所有表面浓度下对总体底物浓度均表现出S形动力学。最后，我们表明磷脂酰丝氨酸（PS）和磷脂酸（PA）可显著增强DGKA的催化活性。
