Second Place


CHEM 435

Publication Date

Fall 2019


Enzymes and Coenzymes | Medicinal and Pharmaceutical Chemistry | Pharmaceutical Preparations | Respiratory Tract Diseases

Description or Abstract

In a year, Tuberculosis (TB) caused approximately a million deaths in addition to 10 million infection cases; thus, it has been subjected to intensive study with the goal of creating a novel therapeutic drug – specifically allosteric inhibitors. Mycobacterium tuberculosis, a pathogenic bacteria that causes TB, has an enzyme found in all organisms and is involved in essential metabolic reactions within the cell, known as Malate Dehydrogenase (MDH). Understanding the structure and function relationships in MDH as well as the specific steps of catalysis can assist in biotech purposes by designing a drug that can specifically bind to a region of pathogenic MDH. MDH catalyzes the reversible conversion of malate to oxaloacetate coupled with the concomitant co-factor conversion of NAD+ to NADH. It is regulated via allosteric inhibition by citrate and substrate inhibition by oxaloacetate. The homodimeric enzyme contains an active site and within this active site there is a mobile loop (P119-N137) that exhibits an opened conformation when substrate/co-factor is bound and a closed conformation when none is bound. Two out of the three catalytic Arg (R124 and R130) is within the loop and its charges play a role in positioning the substrate into the correct orientation for efficient catalysis. To further garner information about the loop, comparison to Lactate Dehydrogenase has been made since it is thought that LDH may have evolved from MDH, a step requiring altering substrate affinity for lactate/pyruvate. Previous studies mutated the catalytic Arg in MDH and revealed an altered substrate affinity to pyruvate rather than oxaloacetate – revealing the importance of the loop’s role in substrate specificity.

To investigate a possible target within the loop that may render pathogenic bacteria MDH inactive without affecting host MDH, sequence and structural bioinformatics have been done to identify residues that are different in Mtb enzyme than the human mitochondrial and cytosolic forms. From sequence alignment of several forms of MDH and LDH, K125 was found to be conserved throughout except in LDH, halophiles and prokaryotic MDH. From HINT computational analyses, it was clear K125 made essential interactions with co-factor and nearby residues that may be involved in catalysis, binding and substrate specificity and several interactions not found in the analysis were predicted. It is hypothesized that the role of K125 may involve the positioning of R124 via charge repulsion and a stabilizing hydrogen bond with G263 to guide the substrate into the active site, thus completing the electrostatic preorganization required for catalysis. To investigate the specific role of K125 in binding, catalysis and specificity, we have constructed via Quikchange mutagenesis as well as expressed and purified via Ni-NTA affinity chromatography two watermelon glyoxysomal MDH (wgMDH) mutants, K125A and K125R. The purified mutants were characterized via Michaelis-Menten enzyme kinetics to determine overall catalytic efficiency as well as substrate and co-factor binding compared to native wgMDH. Substrate inhibition assays were performed utilizing malonate, pyruvate and citrate to assess substrate specificity changes, if any, in the mutants.

Fluorescence-based Thermal Shift Assays (FbTSA) were done to assess changes in stability and co-factor binding of the mutants. Kinetic studies revealed no significant changes in Km of NADH and OAA and changes in Vmax of NADH and OAA, suggesting K125 may not be involved in co-factor/substrate binding. Citrate inhibition kinetics indicated the loss of affinity for citrate in K125A and Malonate inhibition kinetics showed K125R had a slightly higher affinity for malonate compared to native and K125A. This indicates that K125 plays a role in substrate specificity. FbTSA analysis indicated an increase in structural stability for both K125A and K125R compared to native, suggesting it has a role in stability. Thus, it can be proposed from these results that K125 is essential for catalysis by playing a role in substrate specificity and interactions K125 makes with nearby residues are essential for structural stability of MDH. Overall, these results not only contributes to the gaps in knowledge in regards to structure-function relationships of essential enzymes, these findings may also be utilized for biotech purposes. The new discoveries made may provide further insight into potential residual targets within MDH for allosteric inhibitor drugs that can render pathogenic bacterial MDH inactive without the drug being detrimental to the host.