MGDG Synthase of Spinach Chloroplast Envelope: Properties of the Substrate Binding Sites

Abstract

Kinetic properties of the delipidated monogalactosyldiacylglycerol (MGDG) synthase with respect to the various diacylglycerol molecules were first investigated in mixed micelles containing the partially purified enzyme, the substrate (diacylglycerol) and the detergent, according to the “surface dilution” kinetic model proposed by [1]. We concluded that MGDG synthase has different affinities for the diacylglycerol species tested: a lower affinity for C18:0/C18:0, C16:0/C16:0, C16:0/C18:l, C18:3/C18:3 (Km from 0.040 to 0.066 mol-fraction), a higher affinity for C16:0/C18:2, C18:l/C16:0, C18:0/C18:l, C18:l/C18:l (Km values from 0.024 to 0.031 mol-fraction), and an even higher affinity for C18:2/C18:2 with Km of 0.0089 mol-fraction. To provide further evidence for a physiological significance of these data, enzymatic assays were performed within envelope vesicles prepared from thermolysin-treated intact chloroplasts. Kinetic experiments suggested that MGDG synthase possesses distinct and independant binding sites for each substrate, UDP-galactose or diacylglycerol. This was verified by studying pattern of inhibition of MGDG synthase activity by UDP relatively to each substrate. Moreover, protection of MGDG synthase activity against citraconic anhydride (a specific reagent of lysines) by incubating with either UDP-gal or 1,2-diacylglycerol demonstrates that substrate binding is not ordered.