Abstract
Malate dehydrogenases (MDH) and lactate dehydrogenases (LDH) are essential metabolic enzymes that share similar folds and mechanisms despite their strong substrate specificity. The unicellular eukaryotes of the Apicomplexa phylum, responsible for a variety of human diseases, have convergently evolved a unique and highly specific LDH from an MDH. The Apicomplexan LDH and MDH are an excellent model for the evolution of enzyme specificity due to the presence of a putative promiscuous intermediate in the evolutionary pathway. A six amino acid insertion into the active site conferred pyruvate activity by shifting the key catalytic residue from an arginine at position 102 in MDHs to a tryptophan at position 107f. However, the bifunctional intermediate contains both R102 and W107f. Though only one conformation is observed in x-ray crystallography data, I hypothesize that there are two active conformations of the intermediate. I used heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) to visualize a dimer of the promiscuous intermediate in the presence of each substrate. Loss of the tryptophan peak was seen in the presence of pyruvate at 25°C. I used intrinsic tryptophan fluorescence to monitor the catalytic tryptophan in the presence of each substrate. Pyruvate amplifies tryptophan fluorescence, but oxaloacetate demonstrates no\r consistent trend. Mutation of Trp107f to an alanine results in the loss of LDH activity but does not affect MDH activity.