A single serine to alanine substitution decreases bicarbonate affinity of phosphoenolpyruvate carboxylase in C(4)Flaveria trinervia

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the first committed step of C(4) photosynthesis generating oxaloacetate from bicarbonate (HCO(3)(−)) and PEP. It is hypothesized that PEPc affinity for HCO(3)(−) has undergone selective pressure for a lower K(HCO3) (K(m) for HCO(3)(−)) to increase the carbon flux entering the C(4) cycle, particularly during conditions that limit CO(2) availability. However, the decrease in K(HCO3) has been hypothesized to cause an unavoidable increase in K(PEP) (K(m) for PEP). Therefore, the amino acid residue S774 in the C(4) enzyme, which has been shown to increase K(PEP), should lead to a decrease in K(HCO3). Several studies reported the effect S774 has on K(PEP); however, the influence of this amino acid substitution on K(HCO3) has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the K(HCO3) of the photosynthetic PEPc from the C(4)Flaveria trinervia and the non-photosynthetic PEPc from the C(3)F. pringlei. The cDNAs for these enzymes were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain. Our work in comparison with previous reports suggests that K(HCO3) and K(PEP) are linked by specific amino acids, such as S774; however, these kinetic parameters respond differently to the tested allosteric regulators, malate and glucose-6-phosphate.