Open AccessSynergism Between Coenzyme and Carboxylate Binding to Liver Alcohol Dehydrogenase
Author(s) -
ANDERSSON Pia,
KVASSMAN Jan,
OLDÉN Bertil,
PEJTERSSON Göata
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb05493.x
Subject(s) - carboxylate , cofactor , chemistry , nad+ kinase , alcohol dehydrogenase , stereochemistry , enzyme , protonation , binding site , ligand (biochemistry) , zinc , nicotinamide , biochemistry , organic chemistry , receptor , ion
1 The interaction of decanoate and benzoate with the substrate‐binding site in liver alcohol dehydrogenase has been characterized by fluonmetric equilibrium binding studies, using auraimne O as a reporter ligand. 2 The affinity of the enzyme for the carboxylates examined decreases about 50‐fold on complex formation with NADH. The coenzyme‐competitive inhibitors ADP‐ribose and Pt(CN) 2− 4 have a similar destabilizing effect on decanoate binding, indicating that the effect of NADH derives from its negatively charged pyrophosphate group. 3 Carboxylate binding to free enzyme requires the protonated form of an ionizing enzymic group with p K a 9.2, while there is no corresponding effect of pH on binary complex formation with auramine O. Carboxylate binding to the enzyme · NADH + complex exhibits no significant dependence on pH over the pH range 7–10. 4 These results, combined with previously reported data for decanoate binding to the enzyme · NAD + complex, provide clear evidence that carboxylates (in contrast to auramine O) are bound at the active‐site zinc ion of the enzyme in a process regulated by the ionization state of zinc‐bound water. The synergistic effect of NADH and NAD + on carboxylate binding can be qualitatively and quantitatively explained in terms of electrostatic interactions analogous to those proposed to account for the effect of coenzymes on the p K a of zinc‐bound water.