Charge balance in the α‐hydroxyacid dehydrogenase vacuole: An acid test

Abstract

The proposal that the active site vacuole of NAD⁺-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140–Asp mutant enzyme has the same k_cat as the wild type (Asn 140) at low pH (4.5), and at higher pH the K_m for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a K_m that is over 1,000 times that of the K_m of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pK_a of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98–110), which normally seals off the internal active site vacuole from external bulk solvent.