Differences in the interactions of liver alcohol dehydrogenases with probes binding into the substrate pocket

The interactions of three groups of probes (berberine alkaloids, tricyclic psychopharmaca and acridine derivatives) with isoenzymes of horse liver alcohol dehydrogenase and with rat liver alcohol dehydrogenase have been examined. These compounds inhibit the activity of the EE isoenzyme of horse liver alcohol dehydrogenase but differ in their behaviour towards the steroid-active enzymes (i.e. the ES isoenzyme of horse liver alcohol dehydrognase and alcohol dehydrogenase from rat liver): psychopharmaca inhibit, acridines activate and berberines do not bind. The ligands differ also in their influence on the modification of the EE isoenzyme by iodoacetate. Polarities (expressed as Kosower's Z values) of the respective binding sites on the EE isoenzyme were estimated from optical properties of bound probes. Berberines bind into a very hydrophobic area of the enzyme molecule, the binding site for psychopharmaca is moderately hydrophobic and that for acridines is rather polar. Steric arrangements of the binding sites are also discussed. The data presented confirm the existence of three distinct binding sites for these ligands in the substrate pocket of liver alcohol dehydrogenase.     

Protection of substrate against enzymatic action by binding to proteins. Dependence upon enzyme and binding protein

In fetal or adult rats estradiol is carried in the plasma by alpha-fetoprotein or albumin. The protection of the carriers toward enzymatic oxidation by 17 beta-hydroxysteroid dehydrogenase from rat liver has been studied. Concentrations of carrier protein and estradiol were adjusted to give free estradiol concentrations varying from Km/10 to Km/100 and the ratio of the catalytic velocity to that observed for the same concentration of free estradiol in the absence of carrier protein were recorded. With alpha-fetoprotein the ratio fell as expected as the carrier concentration was increased, but with serum albumin the ratio was close to unity when the concentration of carrier protein was increased from 70 to 700 microM. Thus, a fetoprotein but not albumin protected the steroid against catalytic oxidation. Similar experiments were carried out replacing the mammalian enzyme by the dehydrogenase from Pseudomonas testosteroni, and in this case, both carrier proteins protected the substrate. The lack of protection by albumin against the dehydrogenation by the mammalian enzyme is discussed.     

Genetic relationships between the multiple alcohol dehydrogenases of maize

There are three electrophoretically separable sets of alcohol dehydrogenase isozymes in maize. Previous work has shown that two of these isozymes (Sets I and II) share a subunit in common, since mutations in one of the Adh genes, Adh ₁, alter both isozymes. A mutation in the second Adh gene, Adh ₂, has now been induced and recovered. This mutant allele also alters two of the three isozymes—Sets III and II. Adh ₁ and Adh ₂ appear to segregate independently. Gel filtration data show that all ADH isozymes are indistinguishable in size. These findings support the hypothesis that the two Adh genes specify promoters which homo- and heterodimerize, yielding three types of ADH isozymes.This research was supported by National Science Foundation Grant GB 25594. M.F. is a recipient of Public Health Service Genetics Training Grant GM 82-12.     

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD⁺ to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K _d of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD⁺ (e.g., K _d of 87 μM for FurX-NAD⁺). The kinetic data suggest that the four enzymes are efficient “furfural reductases” with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°′) for ethanol-dependent reduction of furfural was determined to be −1.1 kJ mol⁻¹. The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.     

Investigation of the properties of alcohol dehydrogenase from bean,rape, wheat and broad bean

Preparations of alcohol dehydrogenase (ADH) were prepared from germinating seeds by a procedure including fractionation with ammonium sulphate, desalting on a column of Sephadex G-25 and chromatography on DEAE-cellulose, the specific activity of ADH was in the case ofPhaseolus 31 times,Brassica 43 times,Triticum 47 times, andVicia 212 times higher than that of crude extract. The enzymes were homogeneous when filtrated on Sephadex G-200. Molecular weight of all the four studied ADH was approximately 63 000. Some kinetic properties as K_m for ethanol as substrate, substrate specificity towards different alcohols, and the effect of some intermediates of sugar metabolism and of some inhibitors on the activity of the enzymes were also followed. The results obtained are discussed with respect to possible mechanism of action of the plant ADH.     

Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase. Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.     

Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.     

The plant tRNA 3' processing enzyme has a broad substrate spectrum.

To elucidate the minimal substrate for the plant nuclear tRNA 3' processing enzyme, we synthesized a set of tRNA variants, which were subsequently incubated with the nuclear tRNA 3' processing enzyme. Our experiments show that the minimal substrate for the nuclear RNase Z consists of the acceptor stem and T arm. The broad substrate spectrum of the nuclear RNase Z raises the possibility that this enzyme might have additional functions in the nucleus besides tRNA 3' processing. Incubation of tRNA variants with the plant mitochondrial enzyme revealed that the organellar counterpart of the nuclear enzyme has a much narrower substrate spectrum. The mitochondrial RNase Z only tolerates deletion of anticodon and variable arms and only with a drastic reduction in cleavage efficiency, indicating that the mitochondrial activity can only cleave bona fide tRNA substrates efficiently. Both enzymes prefer precursors containing short 3' trailers over extended 3' additional sequences. Determination of cleavage sites showed that the cleavage site is not shifted in any of the tRNA variant precursors.     

Kinetic transients in the reduction of aldehydes catalysed by liver alcohol dehydrogenase.

The transient-state kinetics of enzymic reduction of acetaldehyde and benzaldehyde by NADH, catalyzed by horse liver alcohol dehydrogenase, have been examined under single-turnover conditions, obtained by carrying out reactions either with limiting amounts of enzyme in the presence of 20 mM pyrazole or with limiting amounts of substrate. Analysis of the variation with substrate, coenzyme, and enzyme concentrations of amplitudes and time constants for the exponential transients observed at 328 nm and 300 nm shows that the kinetics of enzymic aldehyde reduction are qualitatively and quantitatively consistent with the relationships derived in the preceding paper for an ordered ternary-complex mechanism involving identical and independent catalytic sites. It is concluded that there is no evidence whatsoever for the kinetic significance of a half-of-the-sites reactivity or any other kind of subunit interaction in the liver alcohol dehydrogenase system. The biphasic transients observed at 328 nm for the reduction of aromatic aldehydes such as benzaldehyde are a normal kinetic characteristic of the ordered ternary-complex mechanism, being attributable to accumulation of the ternary enzyme-NAD-product complex when product dissociation from this complex is slow in comparison to its formation by ternary-complex interconversion.     

Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme

Platelet-activating factor acetylhydrolases (PAF-AHs) are a group of enzymes that hydrolyze the sn-2 acetyl ester of PAF (phospholipase A(2) activity) but not phospholipids with two long fatty acyl groups. Our previous studies showed that membrane-bound human plasma PAF-AH (pPAF-AH) accesses its substrate only from the aqueous phase, which raises the possibility that this enzyme can hydrolyze a variety of lipid esters that are partially soluble in the aqueous phase. Here we show that pPAF-AH has broad substrate specificity in that it hydrolyzes short-chain diacylglycerols, triacylglycerols, and acetylated alkanols, and displays phospholipase A(1) activity. On the basis of all of the substrate specificity results, it appears that the minimal structural requirement for a good pPAF-AH substrate is the portion of a glyceride derivative that includes an sn-2 ester and a reasonably hydrophobic chain in the position occupied by the sn-1 chain. In vivo, pPAF-AH is bound to high and low density lipoproteins, and we show that the apparent maximal velocity for this enzyme is not influenced by lipoprotein binding and that the enzyme hydrolyzes tributyroylglycerol as well as the recombinant pPAF-AH does. Broad substrate specificity is also observed for the structurally homologous PAF-AH which occurs intracellularly [PAF-AH(II)] as well as for the PAF-AH from the lower eukaryote Physarum polycephalum although pPAF-AH and PAF-AH(II) tolerate the removal of the sn-3 headgroup better than the PAF-AH from P. polycephalum does. In contrast, the intracellular PAF-AH found in mammalian brain [PAF-AH(Ib) alpha 1/alpha 1 and alpha 2/alpha 2 homodimers] is more selectively operative on compounds with a short acetyl chain although this enzyme also displays significant phospholipase A(1) activity.     

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 (K_m 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.