Kinetic analyses of recombinant isoforms of phosphoenolpyruvate carboxylase from Hydrilla verticillata leaves and the impact of substituting a C4-signature serine

Hydrilla verticillata (L.f.) Royle is a single-cell C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell; phosphoenolpyruvate 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 C4-signature Ser. Recombinant proteins were generated and the C3-Ala was replaced by the C4-Ser using site-directed mutagenesis. Kinetic analyses of the recombinant isoforms indicated that rHVPEPC4 had a higher K0.5(PEP) (0.3 mM), more typical of C4 PEPC, despite the lack of a signature Ser. Replacing Ala with Ser in rHVPEPC4 further increased the K0.5(PEP) to 0.78 mM, but reduced the Vmax by 25%. However, a similar Ser substitution in rHVPEPC3 not only increased the K0.5(PEP) from 0.15 to 0.92 mM but also increased the I50(MAL), Vmax and kcat. Thus, with a single amino acid substitution rHVPEPC3 kinetically became similar to a C4 PEPC. In all cases, glucose-6-phosphate lowered the K0.5(PEP) values and reduced malate inhibition. Although, the C4-Ser had an impact, overall the C4 characteristics of HVPEPC4 are conferred by other parts of its primary structure.