Carboxymethylation of horse-liver alcohol dehydrogenase in the crystalline state. The active-site zinc region and general anion-binding site of the enzyme correlated in primary and teritiary structures.
Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)2-/4 , 1,10-phenanthroline, Au(CN)-/2 and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboyxmethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN2-/4 to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN2-/4 and to a lesser extent Au(CN)-/2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identificationof this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.
D000249:Adenosine Monophosphate; D000429:Alcohol Oxidoreductases / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003486:Cyanides; D006736:Horses; D007461:Iodoacetates; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D010984:Platinum; D011485:Protein Binding; D011487:Protein Conformation; D014961:X-Ray Diffraction; D015032:Zinc / Q000032:analysis
D003486:Cyanides; D007461:Iodoacetates; D000249:Adenosine Monophosphate; D010984:Platinum; D000429:Alcohol Oxidoreductases; D015032:Zinc