Regulation of C₄-form Phosphoenolpyruvate Carboxylase (PEPC): Molecular Mechanism of pH-dependent Alteration of Malate Sensitivity

Abstract

PEPC involved in C₄ photosynthesis is subject to an allosteric inhibition by malate. The inhibitor-sensitivity is high at pH7, while very low at pH8. There is no such pH-modulation in E.coli enzyme (EcPEPC). Previous our study had revealed that the flexible loop I (631-651 in maize C₄-form PEPC, ZmPEPC) takes two states in association with allosteric activation and inhibition. In the present study a recombinant ZmPEPC was crystallized and X-ray analyzed. The loop I was revealed to be stabilized through interaction with the plant-specific loop II (124-140) at pH7, while it is mobile at pH 8. Furthermore, protonated H653 in the loop I was speculated to play a key role in this stabilization. The kinetic studies with many mutant enzymes as given below essentially supported the speculation and more detailed mechanisms: ZmPEPC (H653N, H653D, H653K, H653R, D133A/E134A, E140A/D142A, G650A/T652A) and EcPEPC (A590G/A592T, A592G/A594T, here H593 corresponds to H653 of ZmPEPC).