Kinetic mechanism and order of substrate binding for sn‐glycerol‐3‐phosphate acyltransferase from squash (Cucurbita moschata)

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Abstract
sn‐Glycerol‐3‐phosphate acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three‐dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451–453; Turnbull et al. (2001) Structure 9, 347–353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl‐acyl carrier protein (acyl‐ACP) substrates. The reaction proceeds via a compulsory‐ordered ternary complex with acyl‐ACP binding before glycerol‐3‐phosphate. We have also determined that the reaction will proceed with C4:0‐CoA, C6:0‐CoA and C12:0‐ACP substrates, allowing a wider choice of acyl groups for future co‐crystallisation studies.