BINDING OF COENZYME, COENZYME FRAGMENTS, AND INHIBITORS TO NATIVE AND CARBOXYMETHYLATED HORSE LIVER ALCOHOL-DEHYDROGENASE FROM CHLORINE-35 NUCLEAR MAGNETIC-RESONANCE QUADRUPOLE RELAXATION

35Cl nuclear magnetic resonance (NMR) quadrupole relaxation measurements have been performed in order to get a detailed picture of the interaction of coenzyme fragments and coenzyme competitive inhibitors with anion binding sites within the active site region of native and carboxymethylated horse liver alcohol dehydrogenase (LADH). A marked complexity of the anion binding was demonstrated with the existence of at least two anion binding sites within the coenzyme binding domain. Both the magnitude of the 35Cl relaxation rate and its change with added ligand point to anion binding to positively charged amino acid residues and in no case could any anion binding to the Zn atoms be demonstrated. A correlation of the present data with X-ray crystallographic data suggests Arg-47 and Arg-271 to be involved in anion binding. The sequences of increasing affinity among the ligands investigated (inter alia adenosine, adenosine monophosphate, adenosine diphosphate, adenosinediphosphoribose, salicylate, 8-anilino-l-naphthalenesulfonate, NADH) are quite different for the two sites and it was also found that the sites are not independent but show an internal cooperativity. By studying the 35Cl NMR quadrupole relaxation in solutions of LADH in the presence of 1, 10-phenanthroline and either NADH or Pt(CN)42-, it was found that the coenzyme molecule is sterically hindered to bind to the enzyme in the presence of the chelator; this applies not, however, for Pt(CN)42~. Carboxymethylation of Cys-46 liganded to the catalytic zinc has no or little effect on the anion-binding properties of LADH; the modification does not seem to alter the zinc into a strong anion-binding site. By studies of both the longitudinal (T1) and transverse (T2) relaxation times of 35Cl as a function of added coenzyme or another ligand, it could be established that the anion-binding group has a marked and rapid internal mobility. © 1979, American Chemical Society. All rights reserved.