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Pyrazole is a strong inhibitor of liver alcohol dehydrogenase in combination with oxidized coenzyme NAD+. We have studied three different complexes of the inhibitor with the enzyme by using crystallographic methods: (1) the binary complex with pyrazole to 3.2-A resolution, (2) the ternary ternary complex with NAD+-4-iodopyrazole to 2.9-A resolution. Crystals of the binary complex are isomorphous to the apoenzyme, and pyrazole binds to the active-site zinc atom in a way analogous to imidazole. Crystals of the two ternary complexes are isomorphous with the ternary alcohol dehydrogenase-NADH-dimethyl sulfoxide complex. One of the nitrogen atoms of the pyrazole ring is directly bound to the active-site zinc atom with a Zn-N bond distance of 2.1A. The other nitrogen atom is 2 A from the C4 atom of the nicotinamide ring of the coenzyme. The iodine atom in 4-iodopyrazole is located in the hydrophobic substrate cleft. The effect of substitutions on the pyrazole ring are discussed in relation to the structure of the active site and substrate pocket. Pyrazole derivatives with long alkyl chains bound in the 4 position are outstanding inhibitors, and this property is related to the topography of the hydrophobic substrate cleft. The conformation of the oxidized coenzyme in the ternary complexes is essentially the same as that of the reduced coenzyme NADH in the NADH-dimethyl sulfoxide complex.

Horse liver alcohol dehydrogenase (EC 1 .l. 1. l), specifically reconstituted with cobaltous ions in the catalytic metal-binding sites, forms ternary complexes with the chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde and NADH or 1,4,5,6-tetrahydronicotinamide-adenine dinucleotide (H2NADH), in close analogy to the native zinc enzyme [Dunn, M. F. and Hutchison, J. S. (1973) Biochemistry, 12, 4882-48921. Control experiments with enzyme depleted of metal in the catalytic sites demonstrate that presence of a metal ion is an absolute requirement for binding of the substrate. The spectra of both the chromophore and the metal ion are changed significantly upon complex formation. The red shift of the visible absorption band of the substrate molecule is 14 nm larger for both the complexes with NADH and H2NADH as compared to the native zinc enzyme; we take this as evidence for direct coordination of the substrate's carbonyl oxygen to the catalytic cobalt ion. The greatly increased intensity of the metal's visible d-d band upon substrate binding adds support to this conclusion. Since binding of the coenzyme as well as of the analog, which have to precede the binding of the substrate, leads to considerable red shifts of the d-d band at 650 nm (28 nm for NADH and 21 nm for HzNADH), a distortion of the coordination sphere of the catalytic metal ion is triggered prior to substrate binding. Presumably the coenzyme-triggered conformation change of the protein involving the catalytic metal ion results in a modulation of the Lewis acid strength of the metal which then is able to coordinate and activate the substrate.It is well established that two of the four zinc ions in horse liver alcohol dehydrogenase participate directly in the reaction catalyzed by this enzyme [l]. However, the detailed mode of participation of metal ions in the catalytic cycle is unclear and still a matter of debate [2]; apparently the individual steps in this catalysis depend on a subtle interplay between substrate, coenzyme, metal and protein structure. In order to elucidate the chemical nature and the time course of these interdependent events, molecular probes have been introduced into the system, such asAbbreviations. Chromophore, trans-4-(iV,N-dimethylamino)-cinnamaldehyde; H2NADH, 1,4,5,6-tetrahydronicotinamideadenine dinucleotide; in Me(c)Me(n)-enzyme, Me = metal, c = catalytic, n = noncatalytic, enzyme = horse liver alcohol dehydrogenase.Enzyme. Horse liver alcohol dehydrogenase (EC 1.1.1.1).cobaltous ions instead of the native zinc [3,4] and chromophoric substrates [5 -71. In particular, the chromophore trans-4-(N,N-dimethylamino)-cinnamaldehyde serves the dual purpose of being a substrate at neutral pH and forming a stable transient in the complex with the enzyme and NADH at high pH [ 5 ] . In this stable ternary complex, the absorption maximum of the chromophore is redshifted by 66nm. This has been taken as strong evidence for a direct coordination of the substrate's carbonyl group with the catalytic zinc ion, which acts as a Le...

An absorption band at 340 nm is shown to be formed concomitantly with the covalent bond between the affinity label 3-chloroacetylpyridine -adenine dinucleotide (clac3PdAD ') and glyceraldehyde-3-phosphate from sturgeon. This band corresponds to a charge-transfer transition. Its intensity depends upon the pH and the ionic strength but is almost independent of the nature of the anions present in the medium. The pH dependence shows an inflexion point at pH 7.1. This result suggests the participation of a residue with a pK, of 7.1 within the active site of the enzyme in the formation of this transition. Using various techniques, the amino acid alkylated by clac3PdAD+ is shown to be the essential Cys-149, thus excluding the participation of this residue in the formation of the charge-transfer transition. On the other hand, the modification of Cys-153 seems not to affect this charge-transfer band. Other possible donors are proposed, such as the invariant His-176 or Tyr-317 residues. These amino acids might be implicated in the formation of the Racker band.In a previous paper, 3-chloroacetylpyridine -adenine dinucleotide was shown to inactivate glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus and sturgeon muscle via an affinity label mechanism [I]. The labeling of both glyceraldehyde-3-phosphate dehydrogenases was demonstrated to be irreversible, with a covalent incorporation of one mole of inactivator/mole enzyme subunit [I].The pH dependence of the inactivation rate and the results on thiol titration under denaturating conditions were rather indicative of a modification of a cysteine residue [l]. More surprising were the results on thiol titration under native conditions which showed the accessibility of one Cys residue per subunit toward 5,5'-dithiobis(2-nitrobenzoate).This result suggested that the modification of a Cys, presumably Cys-349, could lead to a partial unfolding of the structure of the protein. As a consequence, other thiol residues would then become accessible to thiol reagents.Results presented in this paper show that an absorption band at 340 nm is formed concomitantly with the covalent bond between 3-chloroacetylpyridine -adenine dinucleotide and glyceraldehyde-3-phosphate dehydrogenase from sturgeon. The properties of this new absorption band raised the question of the true nature of the amino acid alkylated by clac3PdADC. For this reason using various techniques but in particular high-performance liquid chromatography (HPLC), we have determined the nature of the modified amino acid.Abbreviations. ac'PdAD ', ac3PdADH, 3-acetylpyridine -adenine dinucleotide, oxidised and reduced forms respectively; clac3PdAD+, clac3PdADH, 3-chloroacetylpyridine -adenine dinucleotide, oxidised and reduced forms respectively; alkylated enzyme, ~-glycerdldehyde-3-phosphate dehydrogenase fully alkylated by clac3PdAD+ ; Tes, 2-{ [2-hydroxy-I ,I -bis(hydroxymeth yl)ethyl]amino}ethanesulfonic acid ; HPLC, high-performance liquid chromatography.Enzymes. D-Glyceraldehyde-3-phosphate dehydrogenase or ...

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