Kinetic analyses of recombinant isoforms of phospho enolpyruvate carboxylase from Hydrilla verticillata leaves and the impact of substituting a C 4-signature serine

Abstract

Hydrilla verticillata (L.f.) Royle is a single-cell C 4 NADP-malic enzyme species in which the C 4 and Calvin cycles operate in the same cell; phospho enolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) is the initial carboxylase. The cDNAs of two Hydrilla PEPC isoforms were sequenced; one encodes the photosynthetic form, HVPEPC4, and the other an anaplerotic, HVPEPC3. Both lack the C 4-signature Ser. Recombinant proteins were generated and the C 3-Ala was replaced by the C 4-Ser using site-directed mutagenesis. Kinetic analyses of the recombinant isoforms indicated that rHVPEPC4 had a higher K 0 .5 (PEP) (0.3 mM), more typical of C 4 PEPC, despite the lack of a signature Ser. Replacing Ala with Ser in rHVPEPC4 further increased the K 0.5(PEP) to 0.78 mM, but reduced the V max by 25%. However, a similar Ser substitution in rHVPEPC3 not only increased the K 0.5(PEP) from 0.15 to 0.92 mM but also increased the I 50(MAL), V max and k cat. Thus, with a single amino acid substitution rHVPEPC3 kinetically became similar to a C 4 PEPC. In all cases, glucose-6-phosphate lowered the K 0.5(PEP) values and reduced malate inhibition. Although, the C 4-Ser had an impact, overall the C 4 characteristics of HVPEPC4 are conferred by other parts of its primary structure.