Regulation and molecular modification of Phosphoenolpyruvate Carboxylase in C4 plants
Creators: name: Gao, Yuan
Publisher: name: Charles Darwin University
Year: 1996

description: The research in this thesis has been concerned with (1) the regulation of maize (monocot C4) and F. trinervia (dicot C4) PEPC by light, protein phosphorylation and metabolite activation; (2) the functional roles of several conserved positively charged amino acid residue of F. trinervia PEPC; and (3) the domains of glycine and glucose 6-phosphate (G6P) activation of maize PEPC. Variations in Ki (malate) values and light activation of C4 PEPC have been reported in the literature. The reasons for many of these observations are not well understood although differences in assay conditions e.g. assay buffer and pH may partly account for the variations observed. My studies in Chapter 2 examined the effect of assay buffers on the light activation of PEPC. The results showed that light activation varied with the type of assay buffer used. Increases of 60-70% were obtained in Hepes, Mops or Tes buffers but not in Tris or Bis-tris buffers. Malate sensitivity of the enzyme also varied with assay buffer. Although phosphorylation decreased and dephosphorylation increased malate inhibition in all the buffers examined. the inhibition was significantly lower in Tris and Bis-tris buffers compared to Hepes, Mops and Tes buffers. The Ki (malate) value in 100 mM Tris buffer was 10-fold higher than in 50 mM Hepes buffer. Hence it is important to select a suitable buffer for studies on PEPC. My results could help to explain the lack of light activation reported in some studies and partially account for the large variations of Ki (malate) values reported in the literature. It has been proposed that the regulation of PEPC during C4 photosynthesis is mediated via protein phosphorylation on a serine residue near the N-terminus of the polypeptide. The primary role of phosphorylation is to activate and protect the enzyme against malate inhibition. However, the observed increase in Ki (malate) value is an order of magnitude smaller than the cellular malate level in illuminated maize leaves. Chapter 3 examines the regulation of maize PEPC by protein phosphorylation and metabolite activation. Glycine (or alanine) and G6P stimulated catalytic activity and increased the affinity for PEP by factors of 8 and 4 respectively. These metabolites stimulated activity more strongly at lower than higher pH and dramatically changed the pH response of the enzyme. Activity increased between pH 6.8 and 8.0 by 10 fold in the absence and only 26% in the presence of these metabolites. In vitro phosphorylation of PEPC increased the activity 2 fold in the absence but not the presence of these metabolites. Malate was a strong inhibitor of PEPC and the Ki (malate) value determined was 0.25-0.5 mM. Protein phosphorylation and the above metabolites increased the Ki value by factors of 3 and 12 respectively, but together they synergistically increased the Ki value 50-fold. Hence maximal protection against malate inhibition was only observed in the phosphorylated enzyme in the presence of metabolites. Furthermore, PEPC activity comparable to the photosynthetic rate measured in intact leaves was obtained only in the presence of metabolites and physiological concentration of malate (20 mM) in crude extract of light-adapted but not dark-adapted leaves. Evidently both light-induced protein phosphorylation and metabolic activators are involved in PEPC activation. I propose that both factors act synergistically to modulate PEPC during photosynthesis in maize leaves. The majority of studies on C4 PEPC are done with monocot species. Relatively little is known about the regulation of PEPC in dicot c4 plants. Chapter 4 examines PEPC regulation in the C4 dicot Flaveria trinervia. Unlike the maize enzyme, flaveria PEPC was not activated by glycine, alanine or G6P and was weakly activated by light. The light-adapted enzyme was relatively insensitive to malate inhibition with a Ki value of 9.4 mM at pH 7.3, mice the value obtained for the dark-adapted enzyme. Decreased malate sensitivity in the light enzyme seemed to compensate for the lack of metabolite activation observed. Alkaline phosphatase decreased the activity of the purified light enzyme significantly and the treated enzyme had properties similar to those of the dark-adapted enzyme with respect to malate sensitivity and catalytic activity. The results suggest that light regulation of PEPC also involves protein phosphorylation. However, I was unable to phosphorylate the purified dark enzyme (despite the addition of protease and phosphatase inhibitors during the extraction and purification) by cAMP-dependent protein Kinase nor was I able to label the protein in vivo by feeding 32Pi to detached leaves. I could find no report on the phosphorylation of dicot C4 PEPC in the literature. In contrast, phosphorylation has been observed for PEPC from a number of dicot C3 and monocot C3 and C4 species. However, the absence of evidence in dicot C4 PEPC might be attributed to the loss of N-terminal region during enzyme purification and it is conceivable that protein phosphorylation may still play a role in PEPC regulation in leaves of F. trinervia. Chapter 5 examined the functional roles of several conserved positively charged amino acid residues (Lys600, Lys829, Arg450, Arg767) in F. trinervia PEPC. A full-length PEPC cDNA which encodes a functional enzyme was cloned into the ppc mutant E. coli strain PCR1. Conversion of Lys600 to Thr600 by site directed mutagenesis increased the Km (PEP) and Km (Mg2+) 3 fold and Km(HC03) 10 fold indicating that the Lys600 residue is likely to be involved in the binding of HC03. Conversion of Lys829 to Gly829 increased the Km (PEP) 1-2 fold at pH 7.3 and 8.0 and the Km (Mg2+) 12 fold at pH 8.0 and 2 fold at pH 7.3. Thus Lys829 might be associated with the PEP and/or Mg2+ binding domain. However, the results indicated that both lysine residues were not obligatory for the catalytic activity which is different from results obtained in studies of chemical modifications. Both the mutant Gly450 and Gly767 PEPC proteins were similarly expressed as the wild-type protein in E. coli PCR1. However, the mutant enzymes did not complement the glutamate requirement of E. coli strain PCR1 and hence could not grow in minimum medium suggesting that the expressed proteins were non-functional in the host cell. The partially purified mutant enzymes had no detectable PEPC activity. Therefore, Arg450 and Arg767 are essential for PEPC function but their precise roles in catalysis remained to be determined. The maize enzyme contains G6P and glycine activation sites whereas the protein from F. trinervia is insensitive to both glycine and G6P activation. Chimeric proteins consisting of maize and F. trinervia PEPC components were used to identify the putative domains of G6P and glycine activation of maize PEPC in Chapter 6. Five chimerics were constructed but only two, ZFB and FZBE, were found to be catalytically active. Both were activated by G6P but not glycine. They both contain the amino acid residue 193-359 region from maize PEPC indicating that the putative G6P domain in maize PEPC is at least partially located within this region. The first 193 N-terminal amino acids in ZFB is derived from F. trinervia while the remainder of the polypeptide is of maize origin. Thus this N-terminal region could conceivably be associated with glycine activation. However, further studies are required to substantiate and confirm the role of this putative glycine domain. In summary, maize but not F. trinervia PEPC is regulated by a dual system of protein phosphorylation and metabolite activation during C4 photosynthesis. The putative domains for glycine and G6P activation in maize PEPC are located in the N-terminal region of the polypeptide while Lys600, the bicarbonate binding site, and Lys829 which is associated with the PEP and/or Mg2+ domain lie in the C-terminal region of the F. trinervia polypeptide. None of these two conserved lysine residue is obligatory for PEPC function but Arg450 and Arg767 are essential for catalytic activity in the flaveria enzyme.