A single serine to alanine substitution decreases bicarbonate affinity of phospho enol pyruvate carboxylase in C 4 Flaveria trinervia

Abstract

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 HCO 3 (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 HCO 3 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 HCO 3 . Several studies reported the effect S774 has on K PEP; however, the influence of this amino acid substitution on K HCO 3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the K HCO 3 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 HCO 3 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.