The Role of the Flexible Loop in Substrate Recognition and Catalysis in Malate Dehydrogenase

Abstract

All enzymes do three things: they bind a specific substrate, catalyze a reaction, and release the product. In addition to providing insight into structure‐function relationships, understanding the molecular basis of steps involved in substrate binding can provide insight into 1.) how to design specific ligands to inhibit, or 2.) how to manipulate substrate specificity to be used for biotech purposes. Malate dehydrogenase (MDH), present in all organisms, plays critical metabolic roles in the cell and catalyzes the interconversion of malate and oxaloacetate. Within the active site of MDH, there is a flexible loop (P119‐N137) that contains two of three arginines whose positive charge interact with the negatively charged 4‐carbon dicarboxylic acid substrate to coordinate its orientation for catalysis. In LDH, (which evolved from MDH) there is a similar flexible loop region which binds pyruvate (a 3‐carbon mono carboxylic acid), Previous work with Ecoli MDH, creation of an R124Q mutation in the flexible loop, led to a change in substrate specificity and also an increased affinity for pyruvate, supporting the suggestion that the flexible loop of MDH is important in discrimination between substrates. Citrate, a 6 carbon tricarboxylic acid has been shown to bind to the active site of MDH in a half‐sites manner, and is thought to regulate activity in some MDHs. We hypothesize that other residues in the loop region are involved in fine tuning both binding and catalysis. To explore this hypothesis we have used watermelon glyoxysomal MDH (WMgMDH) as a model and constructed a variety of mutations throughout the loop to explore how the loop is involved in both discriminating between ligands (including substrate, citrate, and pyruvate) and contributing to catalysis. To explore potential interactions that residues in the loop makes, HINT computational analysis was used to predict the interactions individual residues make with the rest of the protein or substrates. A series of site‐directed mutants were created: A120G, G121A, P123S, F134S, D132A, L133S, F134S, and N137A. using Quikchange mutagenesis. Mutants were expressed and purified using Ni‐NTA affinity chromatography and characterized. Michaelis Menten Kinetic studies were used to determine overall catalytic efficiency, substrate binding and specificity, and cofactor binding, and compared to wild‐type. D132A, L133S, F134S, and N137A had a significant decrease in specific activity. A120G, G121A, L133S, and N137A showed anomalous NADH saturation. G121A, P123S, M128A, D132A, L133S, F134S, and N137A showed significant changes in oxaloacetate binding. An interesting trend for the four mutants that were farther away from R124 and R130 (D132A, L133S, F134S, and N137A) was that those that had the highest affinity for NADH had the lowest affinity for OAA and vice versa. These results suggest that the “specificity loop” of Malate Dehydrogenase plays additional roles in cofactor binding and catalysis than previously thought, and could provide new insights that could be exploited for biotech purposes as well as furthering our knowledge of structure‐function relationships in this important metabolic enzyme.Support or Funding InformationThis work was supported by NSF Grants 1726932 and 0448905