Enzymes rely on a highly conserved active site sequence in order to maintain activity towards and specificity for their substrates. This is particularly important in the case of cytoplasmic enzymes such as malate dehydrogenase (MDH), which will encounter many molecules which closely resemble their intended substrate. However, not every amino acid that is important in regulating enzymatic activity interacts directly with the substrate. Two point mutants of MDH (R81W and R81N) were utilized in an attempt to better understand the role that Arginine<sup>81</sup> plays in regulating the catalytic activity of MDH. Contrary to the hypothesized result, R81W displayed a significant (p=0.002) increase in specific activity towards malate from the wild type MDH (WT). The R81N mutant was not isolated sufficiently to quantify specific activity, however it showed considerably less activity (about a 60% decrease from WT) despite SDS-PAGE indications that there was considerably more of the mutant enzyme present. Thus, Arginine<sup>81</sup> plays a significant role in regulating the catalytic activity of MDH, which can likely be attributed more to its steric properties than its electrostatic properties.

Utilizing point mutants of malate dehydrogenase to elucidate the role of arginine 81 in catalytic specificity

ACS Spring 2022

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