This project focuses on the steady-state enzyme kinetics of mammalian L-lactate dehydrogenase (LDH) in a crowded macromolecular environment. LDH is a cytosolic enzyme that catalyzes the reduction of pyruvate to L-lactate and the oxidation of NADH to NAD+ as the final step in lactic acid fermentation. Bovine serum albumin (BSA), a soluble globular protein, was used as the primary model crowder. Using NADH as variable substrate, LDH was demonstrated to exhibit positive cooperativity with a Hill coefficient of 2.5 in the absence of crowder. Addition of increasing concentrations of BSA resulted in progressively decreasing Hill coefficients and concomitant increases in K0.5 and Vmax. No cooperativity was observed with pyruvate as variable substrate, but increases in BSA concentration did increase Km and Vmax. In the reverse reaction of lactate to pyruvate, no cooperativity was observed with either NAD+ or L-lactate as variable substrate in either the absence or presence of macromolecular crowder. With NAD+ as variable substrate, increasing concentrations of BSA did not significantly change Vmax or Km. With L-lactate as variable substrate, Km and Vmax showed non-linear trends as a function of crowder concentration. Our current aim is to determine crowding effects on oxamate inhibition of LDH for both forward and reverse reactions.

Macromolecular crowding effects on Lactate Dehydrogenase (LDH)

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.