Investigations of enzyme active-site architectures and catalysis

Abstract

Sodium methyl hex-5-ynoyl phosphate (SMHP) was synthesized as an activity-based probe designed to target protein architectures with nucleophiles adjacent to a cationic binding site. Activity-based protein profiling revealed that SMHP modified 281 enzymes from the cell-lysate of Pseudomonas lemognei, including D-3-hydroxybutyrate dehydrogenase and CTP synthase. The sites of modification were investigated using fluorescence-based activity studies, LC-MS/MS, and kinetics. The inactivation of L-fuconate dehydratase (FucD) by 3-hydroxypyruvate and the effects of Tris buffer were also explored. With increasing Tris concentrations, the kinactapp and KIapp values decreased, but the kinactapp/KIapp remained unchanged (~0.018 ± 0.002 M–1s–1). Finally, the Cys to Ser variants (C76S and C186S) of glutamate racemase from Fusobacterium nucleatum were constructed. The C76S variant exhibited greater catalytic efficiency turning over D-Glu relative to L-Glu at higher pH values; however, this preferred ‘unidirectional’ behavior was not observed for the C186S variant in the L-Glu to D-Glu reaction direction.