Effects of Bilayer Thickness on the Activity of Diacylglycerol Kinase of Escherichia coli

Abstract

We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate. When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine [di(C18:1)PC] and a lower activity in bilayers with shorter or longer fatty acyl chains. Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine [di(C14:1)PC] followed from an increase in the K_m value for DHG and ATP, with no effect on v_max. In contrast, in the long chain lipid dierucoylphosphatidylcholine [di(C24:1)PC], the low activity followed from a decrease in v_max with no effect on K_m. In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.