Biochemical studies on plant glycerol-3- phosphate acyltransferase
sn-Glycerol-3-phosphate acyltransferase [G3PAT, PlsB (E.coli), EC 22.214.171.124] is an enzyme involved in glycerolipid biosynthesis, catalysing the acylation of glycerol-3-phosphate (G3P) to produce lysophosphatidic acid (LPA). Chilling tolerance in plants is linked to the acyl-group composition of membranes, which is linked to acyltransferases with a higher selectivity for unsaturated acyl-substrates. Plant soluble G3PAT is located in the chloroplast and uses acyl-acyl carrier protein (acyl-ACP) as substrate. Soluble G3PAT exhibits strong substrate selectivity for acyl-ACP, the plastidial substrate in vivo, over acyl-CoA. cDNAs encoding soluble G3PATs have previously been cloned from several plant species and both oleate-selective and non-selective forms identified. The purpose of this thesis is to study the mechanism of plastidial G3PAT and attempt to identify factors important in determining substrate selectivity. An in vitro assay has been optimised to distinguish selective and non-selective enzyme forms under physiologically relevant conditions. The assay has been adapted to determine enzyme activity with a range of acyl-ACP and acyl-CoA substrates and to measure the kinetic constants Km and Vmax. Kinetic measurements have been made on a G3PAT protein from the chilling sensitive plant squash (Cucurbita moschata) and the L261F mutant protein containing a single amino acid substitution that significantly alters substrate selectivity. The mutation was found to increase selectivity by raising Km for unsaturated acyl-substrate. Mutant squash G3PAT proteins have been investigated to determine the importance of particular regions or amino acid residues. The mutations E142A, K193S, R235S and R237S resulted in enzymes that were completely inactive. The mutations H194S and L261F altered catalytic or substrate binding characteristics without enzyme inactivation. The catalytic mechanism and order of substrate binding for squash G3PAT have been determined, the reaction was found to proceed via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate.