Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase

Abstract

The pH-dependencies of the binding of NADH and reduced nicotinamide-benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase [EC 1.1.1.34] and lactate dehydrogenase [EC 1.1.1.27] are reported. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3-6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165. Only 1 ionization is required to characterize the binding of the 2 reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4-7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. Two reactive histidine residues were detected per malate dehydrogenase dimer. A mechanism which emphasizes the homology between the 2 enzymes is presented.