Regina Pietruszko

Comparison of substrate specificity of alcohol dehydrogenases from human liver, horse liver, and yeast towards saturated and 2-enoic alcohols and aldehydes

The saturated and 2-enoic primary alcohols and aldehydes, ethanol, 1-propanol, 1-butanol, 3-methyl-1-butanol, 1-hexanol, phenylmethanol, 3-phenyl-1-propanol, 2-propen-1-ol, 2-buten-1-ol, 3-methyl-2-buten-1-ol, 2-hexen-1-ol, 3-phenyl-2-propen-1-ol, ethanal, 1-propanal, 1-butanal, 1-hexanal, phenylmethanal, 3-phenyl-1-propanal, 2-propen-1-al, 2-buten-1-al, 2-hexen-1-al, and 3-phenyl-2-propen-1-al, have been compared under uniform conditions as substrates for the alcohol dehydrogenase enzymes from horse and human liver and from yeast. Kinetic constants (Km arid V) have been measured for each of the substrates with each of the enzymes; equilibrium constants for the various alcohol-aldehyde pairs have also been estimated. The results obtained emphasize the similarities of yeast alcohol dehydrogenase to horse and human liver alcohol dehydrogenase, showing the specificity of yeast alcohol dehydrogenase to be less restricted than formerly believed. In general, the 2-enoic alcohols are better substrates for all three alcohol dehydrogenases than their saturated analogs; on the other hand, saturated aldehydes are better substrates than the 2-enoic aldehydes. Based on these various findings, it is suggested that a more likely candidate than ethanol for the physiological substrate of alcohol dehydrogenase in mammalian systems may well be an unsaturated alcohol, although the wide variety of substrates catalyzed at high rates is not incompatible with a general detoxifying function for alcohols or aldehydes, or both, by alcohol dehydrogenase.

Human liver alcohol dehydrogenase--inhibition of methanol activity by pyrazole, 4-methylpyrazole, 4-hydroxymethylpyrazole and 4-carboxypyrazole.

Abstract With human liver alcohol dehydrogenase of high purity at pH 7.0 and 500 μM NAD the Km for methanol is 7.0 mM (ten times greater than the Km for ethanol) and the turnover number 1.4/active site/min (about one-tenth of the turnover with ethanol in the same conditions). From secondary kinetic plots it can be calculated that at saturating concentrations of both substrates, namely methanol and NAD, these constants do not change appreciably: the Km for methanol is somewhat lower (5.2 mM) and the turnover number slightly higher (1.7/active site/min). The difference in turnover numbers with methanol and ethanol as substrates suggests that the kinetic mechanism for methanol is different from that for ethanol dehydrogenation. The dissociation constant between human alcohol dehydrogenase and NAD, determined kinetically with methanol as substrate, is 127 μM. The Ki values for pyrazole, 4-methylpyrazole and 4-hydroxymethylpyrazole are 0.54, 0.09 and 6.6 μM respectively; 4-carboxypyrazole (100 μM) at 3mM methanol does not inhibit human ADH. The inhibitory effect of 4-methylpyrazole is therefore not likely to be enhanced by a possible metabolic conversion to 4-hydroxymethylpyrazole and 4-carboxypyrazole.

Methanol activity of alcohol dehydrogenases from human liver, horse liver, and yeast☆

Abstract Formaldehyde and NADH are formed from methanol and NAD in the reaction catalyzed by horse liver ADH and yeast ADH. In this reaction the above enzymes resemble closely human liver ADH, which has been known for some time to catalyze methanol dehydrogenation. The formation of formaldehyde from melhanol has been followed by chromatography of 2:4-DNPH derivatives of the reaction products and quantitation by the highly specific chromotropic acid method. Kinetic results have shown that turnover numbers × sec −1 × active site −1 are very similar for all three enzymes. The specificity of all three enzymes with respect to methanol is therefore identical. Human liver ADH, however, has the lowest K m for methanol, and on this basis only may be considered to be the best catalyst of this reaction.

Mammalian liver alcohol dehydrogenases.

Literature on the properties of liver alcohol dehydrogenase (ADH) from man, horse and rat is reviewed and discussed under two major headings: 1) physical and chemical properties of ADH and 2) structure-function relationship in isoenzymes. Under the first heading are discussed: molecular weight, subunit composition catalytic sites per molecule, sulfhydryl groups, end groups, amino acid composition, role of Zn++ in the structure and function, coenzyme specificity and binding, conformational changes, substrate specificity, catalytic mechanism and recent results from x-ray crystallography of horse liver ADH. The physicochemical properties of ADH from man, horse and rat are for the most part similar. All three enzymes have identical molecular weights, similar amino acid compositions, consist of two subunits, and are all metalloenzymes containing Zn++: horse and human ADH contain one coenzyme binding site per subunit; no results are available for the rat ADH. ADH catalyses interconversion of a large variety of saturated and unsaturated aliphatic and aromatic alcohols and the corresponding aldehydes and ketones utilizing NAD(H). The physiological role of ADH is uncertain. ADH readily combines with reduced coenzymes to form binary complexes with low dissociation constants (10-7 to 10-8M); in the ternary complexes with coenzymes and substrate-competitive inhibitors, these constants are even lower. In the presence of suitable inhibitors, the enzymes can be titrated by coenzymes employing fluorometric and spectrophotometric procedures. The rate of the overall reaction catalyzed by ADH is determined by the dissociation rates of coenzymes, the slowest steps in the reaction sequence. Under the second heading are discussed: liver ADH isoenzymes of horse, man, rat, rhesus monkey and other species, and the significance of steroid activity which accounts for the distinct substrate specificity of some isoenzymes. ADH from horse liver is a heterogeneous enzyme consisting of subunits of distinct substrate specificity and primary structure. The difference in the amino acid sequence between subunit E (active with classical ADH substrates, but not with steroids) and subunit S (active also with steroids) amounts to six amino acids out of 374. Human ADH is also heterogeneous, and at least five genes code for polypeptides which, by dimerization, form different isoenzymes. Experimental evidence suggests that rat ADH is a single unique protein which, like horse liver ADH, SS, is active with steroids. The physiological significance of steroid activity of ADHs is unknown. (Four tables with comparative data and one figure are presented).

Aldehyde dehydrogenase in human blood

Oxidation of ethanol in the liver leads to the formation of acetaldehyde which is potentially more toxic than ethanol [l] . Korsten et al. [2] have demonstrated that following administration of ethanol, blood acetaldehyde concentrations are elevated to a greater extent in alcoholics (42.7 nmol/ml) than in non-alcoholics (26.5 nmol/ml) which suggests that in alcoholics either the rate of formation or of catabolism of acetaldehyde is altered Thus, acetaldehyde seems to be implicated in alcoholism-related disorders (Korsten et al. and Raskin [2]). This paper is the first report of occurrence of aldehyde dehydrogenase (EC 1.2.1.3) in blood. In the rat high aldehyde dehydrogenase levels in organs other than liver have been found [3] ; no aldehyde dehydrogenase activity was, however, detected in the blood. The disappearance of acetaldehyde upon incubation with rat blood has been attributed to irreversible binding of acetaldehyde to hemoglobin [4]. Recently, two aldehyde dehydrogenases differing greatly in kinetic properties were isolated here from human liver [5] . Using human liver enzymes as markers, samples of blood (the only readily available human tissue) from a number of individuals with no known alcoholic history were examined. The results demonstrating and identifying aldehyde dehydrogenases in human blood are presented in this paper.

Kinetic mechanism of the human cytoplasmic aldehyde dehydrogenase E1

The kinetic mechanism of human liver aldehyde dehydrogenase, E1, was investigated at pH 7.2 using initial velocity, product inhibition, dead end inhibition, and pre-steady-state techniques. Only NADH, which was found to be competitive with NAD+ at high (mm) and low (μm) concentrations of acetaldehyde, could be used as a product inhibitor. Chloral hydrate was found to competitively inhibit the enzyme with respect to acetaldehyde and also produce a hyperbolic slope replot when NAD+ was varied. Adenosine 5′-monophosphate was competitive with NAD+ and noncompetitive with acetaldehyde as the varied substrate. The results indicate that the reaction mechanism proceeds mainly through an enzyme·NAD binary complex, but at high concentrations of aldehyde a small degree of randomness occurs. In order to gain information concerning the rate-limiting step, deuteroacetaldehyde was used and found to increase the Km of the aldehyde (KmCH3CDOCH3CHO=3) but have no effect on the steady-state maximal velocity. When enzyme·NAD was rapidly mixed with acetaldehyde in the stopped-flow apparatus, a “burst” of NADH was observed followed by a slower steady-state turnover, indicating that the rate-limiting step of the dehydrogenase reaction occurs after ternary complex interconversion. The magnitude of the “burst” using high specific activity E1 was that which would be expected for four active sites per tetramer, indicating all-of-the sites reactivity.

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney.

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD+-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD+-dependent retinaldehyde activity was associated with the E1 isozyme (coded for byaldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD+-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD+-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1gene) and broad substrate specificity E1 (a member of EC 1.2.1.3aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.

Heterogeneity of glyceraldehyde-3-phosphate dehydrogenase from human brain

In an attempt to characterize enzymes from human brain capable of dehydrogenating short chain aliphatic aldehydes, four groups of enzymes which catalyze inorganic phosphate-dependent reversible dehydrogenation of glyceraldehyde 3-phosphate as well as short chain aldehydes have been purified and characterized. Three enzyme groups are visualized as multiple bands on isoelectric focusing: E6.6 (pI 6.65, 6.75, 6.85); E6.8 (pI 6.8, 6.9); E8.5 (pI 8.5, 8.6); one enzyme, E9.0, is seen as a single band pI 9.0. The subcellular localization of E6.8, E8.5 and E9.0 appears to be mitochondrial. The mitochondrial enzymes differ slightly in molecular weight: E6.8 is 142,000 with subunits of 36,000 and 38,000; E8.5 is 120,000 with a subunit weight of 29,500; E9.0 is 133,000 with a subunit of 33,000. The E8.5 and E9.0 enzymes also appear to contain Zr as part of their molecular structure. E6.6 (subcellular localization uncertain) is a dimer with a molecular weight of 98,000 and two subunits of 58,000 and 61,000. The specific activity with glyceraldehyde-3-phosphate is: E6.6, 8.6 IU/mg; E6.8, 13 IU/mg; E8.5, 158 IU/mg; E9.0, 620 IU/mg. With glyceraldehyde 3-phosphate and 1,3-diphosphoglyceric acid and Km values of all the enzymes are similar (10-40 microM), except for E6.8 whose Km for glyceraldehyde 3-phosphate is very sensitive to pH and is extremely low at pH 7.0 (2 microM), while being considerably higher than that for the other enzymes at pH 9.0 (170 microM). The molecular properties, Km values as well as high specific activity with glyceraldehyde 3-phosphate identify E6.8, E8.5 and E9.0 as glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.12). The catalytic properties of E6.6 are similar to those of E6.8, E8.5 and E9.0, but its molecular properties are different, precluding definite identification.

Human mitochondrial aldehyde dehydrogenase inhibition by diethyldithiocarbamic acid methanethiol mixed disulfide: a derivative of disulfiram

A derivative and possible physiological metabolite of disulfiram, diethyldithiocarbamic acid methanethiol mixed disulfide, is shown here for the first time to inactivate the mitochondrial low‐K m, isozyme of human aldehyde dehydrogenase (EC 1.2.1.3). By comparing inactivating effects of diethyldithiocarbamic acid mixed disulfides with thiols of increasing chain length evidence is provided that steric hindrance is the reason for lack of inhibition of the mitochondrial enzyme by disulfiram in vitro. Since methanethiol is a normal metabolite [(1983) Annu. Rev. Biochem. 52, 187–222] the results also suggest a mechanism by which aldehyde dehydrogenase is inhibited by disulfiram and diethyldithiocarbamic acid in vivo.

Human mitochondrial aldehyde dehydrogenase substrate specificity: Comparison of esterase with dehydrogenase reaction

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.