Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase.
1. The pH-dependencies of the binding of NADH and reduced nicotinamide--benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. 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-165.3. Only one ionization is required to characterize the binding of the two 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 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.
D001562:Benzimidazoles; D055598:Chemical Phenomena; D002621:Chemistry; D003600:Cytosol / Q000201:enzymology*; D005470:Fluorometry; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism*; D009243:NAD / Q000031:analogs & derivatives; D011485:Protein Binding
D001562:Benzimidazoles; D009243:NAD; D006639:Histidine; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase