The role of the flexible loop region in the substrate binding and catalytic activity of malate dehydrogenases

Malate dehydrogenase (MDH) is a ubiquitous enzyme found across all organisms, playing a central role in cellular metabolism. It catalyzes the interconversion between malate and oxaloacetate (OAA), utilizing NAD(H) or NADP(H) as a cofactor. In this study, we investigated the roles of several amino acid residues within the conserved "flexible loop" region of glyoxysomal MDH from Citrullus lanatus (wgMDH). Specifically, we examined how mutations in this region affect structure, substrate binding, catalysis, and substrate inhibition by OAA. We used kinetic experiments and molecular dynamics simulations to explore these effects. Our results demonstrated the importance of the flexible loop region in positioning the substrate for optimal catalysis, specifically for OAA binding. Several mutants exhibited a significant reduction in affinity for OAA binding while showing minimal effects on NADH binding and little or no decrease in overall catalytic activity. Further, two of the mutations showed decreased K_i for OAA, supporting the impact on OAA binding. Modeling of the effects of the loop on the structure and dynamics of MDH revealed that mutations changed the position of the loop relative to the catalytic histidine, leading to defects in binding and catalysis. These findings suggest that alterations in the dynamics of the flexible loop region influence substrate binding over catalysis.