The mitochondrial isozyme of horse liver aldehyde dehydrogenase was labeled with brominated [5-(3-acetylpyridinio)pentyl]diphosphoadenosine. Specific labeling of a coenzyme binding region was proven by an enzymatic activity of the isozyme with the nonbrominated coenzyme derivative, optical properties of the complex, stoichiometry of incorporation, and protection against inactivation. A cysteine residue was selectively modified by the brominated coenzyme analogue and was identified in a 35-residue tryptic peptide. This cysteine residue corresponds to Cys-302 of the cytoplasmic isozyme and has earlier been implicated in disulfiram binding, confirming a position close to the active site. In contrast, the butyl homologue of the coenzyme analogue labels another residue of the mitochondrial isozyme. Thus, in the same isozyme, two residues are selectively reactive. They are concluded to be close together in the tertiary structure and to be close enough to the coenzyme binding site to be differentially labeled by coenzyme analogues differing only by a single methylene group.

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.

The primary structure of the mitochondrial form of horse liver aldehyde dehydrogenase has been determined, utilizing peptide analyses and homology with other enzyme forms. The subunit exhibits N-terminal heterogeneity in size similar to that for the corresponding human mitochondrial protein, the longest form having 500 residues. Catalase was identified as a contaminant of the preparations. All four pairs within a set of aldehyde dehydrogenases can now be compared, including the same two species variants (horse and human) for both the cytosolic and mitochondrial enzyme, revealing characteristic differences although Cys-302 and other segments of presumed functional importance are unchanged. The cytosolic and mitochondrial enzymes are clearly different (172 exchanges in the horse pair; 160 exchanges in the human pair) and the mitochondrial forms are more conserved (28 exchanges of 500 residues) than the cytosolic ones (43 exchanges). Distributions of the residue substitutions also differ between the two enzyme types. These results suggest a comparatively distant separation of the cytosolic and mitochondrial enzymes into forms with separate functional constraints that are more strict on the mitochondrial than the cytosolic enzyme. Unexpectedly, positions with residues unique to one of the four enzymes are about twice as common in both of the horse proteins than in either of the human proteins. This difference may reflect a general pattern for human/non-human proteins, showing that not only functional properties of the protein, but also other factors, such as generation time (longer in man than in horse), are important for enzyme divergence.

Nicotinamide -5-bromoacetyl-4-methyl-imidazole dinucleotide was synthesized with and without a 32P or I4C label. This NAD analogue acts as hydrogen acceptor during enzymatic oxidation of ethanol by alcohol dehydrogenases. Due to the reactive bromoacetyl group the analogue also inactivates the enzymes by covalent modification of the proteins. Stoichiometry of binding, spectral properties of binary complexes and enzymatic parameters suggest that the analogue is bound at the coenzyme binding sites of the enzymes, where adjacent residues are alkylated.The residues modified in horse liver and yeast alcohol dehydrogenases were identified after coupling with the 32P-labelled analogue, or with the non-radioactive analogue and subsequent reduction with 3H-labelled sodium borohydride. The labelled proteins were digested with chymotrypsin and the radioactive peptides analyzed. One cysteine residue was specifically modified in each of the two proteins. In the yeast enzyme this was Cys-43 in the tentative sequence, while in the horse protein Cys-174 was labelled. Structural work shows that the proteins are homologous and that both cysteine residues (Cys-46 and Cys-174 in the numbering system of the horse protein) are present in both enzymes, at corresponding but numerically slightly different positions. The reagent thus alkylates alternative residues in these related proteins.The results suggest that Cys-46 and Cys-174 are spatially close together at the active site region of horse liver alcohol dehydrogenase, and that the same applies to corresponding residues in the yeast enzyme. This is in excellent agreement with other data from chemical modifications and X-ray crystallographic analyses.Reactive NAD analogues, which bind to the coenzyme binding site of dehydrogenases and covalently modify adjacent amino acids, permit identification of these residues in the primary structure of the protein.In this way spatial conclusions may be obtained, which complement information from other studies in the determination of the structure and function of the protein.

Kinetic investigations employing the substrate analogues 2-oxoglutarate and phospho(enol)pyruvate indicate that the allosteric i*-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei has a non-catalytic pyruvate-binding site to which, in addition to pyruvate, the allosteric effector fructose 1,6-bisphosphate can also be bound.A modification using the ''C-labelled substrate analogue 3-bromopyruvate induces a loss of regulation by fructose 1,6-bisphosphate. The histidine residue labelled by 3-bromopyruvate is homologous to histidine-I 88 which is part of the anion-binding site of the non-allosteric vertebrate L-lactate dehydrogenases.Thus, the allosteric site of the allosteric L-lactate dehydrogenases corresponds to the anion-binding site of the nonallosteric vertebrate enzymes.Primary structure and low resolution crystallographic investigation of the allosteric L-lactate dehydrogenase from Lactobacillus casei [l, 21 indicate that the structure of the bacterial allosteric I>-lactate dehydrogenases is very similar to that of the non-allosteric vertebrate enzymes. However, the structural details responsible for allosteric or non-allosteric behaviour, respectively, have not yet been elucidated.The allosteric enzyme of L. casci exhibits homotropic effects, as shown by sigmoidal pyruvate saturation curves, and heterotropic effects, as shown by the increase of substrate affinity and V induced by Fru(l,6)f2 and Mn2+ ions [3].The homotropic effect can be explained by positive cooperativity of the catalytic pyruvate-binding sites or -assuming additional, non-catalytic, allosteric pyruvate-binding sitesbetween the catalytic and allosteric pyruvate-binding sites. The existence of allosteric pyruvate-binding sites has to be assumed, if substrate analogues can be found, which diminish the cooperativity without competing with pyruvate for the active site. In fact, effects indicating non-catalytic, allosteric pyruvate-binding sites have been found for the allosteric L-lactate dehydrogenases from Streptococcus nzutuns [4] and L. casei [3]. The present paper describes kinetic experiments which give evidence that the allosteric effector Fru(l,6)P2 is also bound to the non-catalytic pyruvate-binding site. In order to label the allosteric site we used the reactive substrate analogue 3-bromopyruvate which can form covalent bond with COTresponding amino acid residues of the protein. This compound proved to be useful in labelling the active site histidine-195 in vertebrate L-lactate dehydrogenase [5]. In this case specific labelling of the active site could be achieved in the presence of the coenzyme, due to the compulsory binding order of the L-lactate dehydrogenases (cf. [6, 71). To avoid, however, an additional labelling of active site residues of the L. caseienzyme we incubated this enzyme in the absence of the coenzyme, a condition under which the active site is not accessible.Abbreviations. DSM, Deutsche Sammlung von Mikroorganismen; Fru(1 ,6)P2, D-fructose 1,6-bisphosphate; HPLC, highperformance liquid chromatography.Enzyme...