Parallelism between ethanol and imidazole interactions with horse liver alcohol dehydrogenase

The rate effects of imidazole on the EE isoenzyme of horse liver alcohol dehydrogenase have been analysed in terms of the elucidated kinetic mechanism of the enzyme. These imidazole effects on both directions of the reaction within nonexcess as well as excess ranges of substrate concentrations pointed to the competition between imidazole and ethanol for binding to the same three enzyme species in the kinetic mechanism, namely the free enzyme, the enzyme-NAD+ complex, and the enzyme-NADH complex. Moreover, both imidazole and ethanol brought about an enhancement in the rate of dissociation of NAD+ from its binding site on the enzyme.     

Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain

The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE.     

Catalysis of the photochemical dismutation of N-methylacridinium cation to N-methylacridone and N-methyl-9, 10-dihydroacridine by hydrophobic sites of horse-liver alcohol dehydrogenase and human serum albumin.

The N-methylacridinium cation is bound to hydrophobic sites of horse liver alcohol dehydrogenase and human serum albumin with an observed stoichiometry of one molecule N-methyl-acridinium chloride per subunit of alcohol dehydrogenase and 2.5 molecules of the dye per molecule human serum albumin; the dissociation constants are 3.6 X 10(-5) M and 1.7 X 10(-5) M, respectively. In light, the proteins catalyze the dismutation of N-methylacridinium chloride to N-methylacridone and N-methyl-9,10-dihydroacridine. The presence or absence of oxygen has no effect upon the observed reaction rate. If horse liver alcohol dehydrogenase is used as catalyst, the reaction is inhibited by adenosine diphosphoribose and by 1,1'-dimethyl-4,4'-bipyridylium dichloride. It is concluded that the N-methylacridinium cation is bound within the catalytic site of the enzyme interacting with the binding sites of the nicotinium ring and/or the binding site of the lipophilic part of the substrate. The anaerobic photodismutation of N-methylacridinium chloride to N-methyl-9,10-dihydroacridine and N-methylacridone can be explained by several alternative patways (see Appendix by S. Hünig), the overall reaction being 2[N-Methylacridinium]+ + H2Ohw leads to N-Methyl-9,10-dihydroacridine + N-methylacridone + 2H+. The prerequisite, a high rate of proton transfer from the reaction site, seems to be common property of the hydrophobic binding regions for the N-methylacridinium cation in both horse liver alcohol dehydrogenase and human serum albumin.     

Ligand discovery using the inter-ligand Overhauser effect: horse liver alcohol dehydrogenase

Two dimensional nuclear Overhauser effect spectroscopy (NOESY) studies on horse liver alcohol dehydrogenase (LADH) in the presence of several ligands revealed unanticipated cross peaks arising from inter-ligand Overhasuer effects (ILOEs) connecting resonances of an inhibitor, m-methylbenzamide, and the reducing agent, cyanoborohydride, initially present to maintain NADH in the reduced state. The presence of NADH was not required to observe of these inter-ligand Overhauser effects. A model for the ternary complex was developed in which the methylbenzamide inhibitors bind to the hydrophobic pocket of the active site involved in benzyl alcohol binding, while the cyanoborohydride coordinates directly with Zn²⁺ at the active site. The observation of these effects supports the use of inter-ligand Overhauser effects for the identification of unanticipated ternary complexes that are of potential utility for the development of novel enzyme inhibitors.     

Separation and partial characterization of multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by O-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5 beta-androstan-3 beta-ol-17-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 microM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.     

Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme

Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges.     

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Inhibition studies of the alleloenzymes AdhS and AdhUF

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melanogaster AdhS and AdhUF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very much as if they were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleolenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of AdhS and AdhUF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295-330 nm. This enables an active site titration to be performed and the determination of epsilon (305 nm) of 15.8 . 10(3) M-1 . cm-1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of the ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and KEI suggests that the coenzyme adenosine binding region is similar is Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in AdhS and threonine in AdhUF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.     

Copper(II)-substituted horse liver alcohol dehydrogenase: structure of the minor species.

Oxygen treatment of horse liver alcohol dehydrogenase EE isozyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of approximately 90% of total Cu(II). The Cu(II) of the remaining 'minor species' cannot be reduced nor does it interact with exogenous ligands, e.g. 2-mercaptoethanol, imidazole, pyrazole, or azide ions. The EPR spectrum is axial with a super-hyperfine splitting of 15.6 G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.     

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.     

Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 delta) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 delta enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-fold increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.     

Characteristics of mammalian class III alcohol dehydrogenases, an enzyme less variable than the traditional liver enzyme of class I

Class III alcohol dehydrogenase, whose activity toward ethanol is negligible, has defined, specific properties and is not just a "variant" of the class I protein, the traditional liver enzyme. The primary structure of the horse class III protein has now been determined, and this allows the comparison of alcohol dehydrogenases from human, horse, and rat for both classes III and I, providing identical triads for both these enzyme types. Many consistent differences between the classes separate the two forms as distinct enzymes with characteristic properties. The mammalian class III enzymes are much less variable in structure than the corresponding typical liver enzymes of class I: there are 35 versus 84 positional differences in these identical three-species sets. The class III and class I subunits contain four versus two tryptophan residues, respectively. This makes the differences in absorbance at 280 nm a characteristic property. There are also 4-6 fewer positive charges in the class III enzymes accounting for their electrophoretic differences. The substrate binding site of class III differs from that of class I by replacements at positions that form the hydrophobic barrel typical for this site. In class III, two to four of these positions contain residues with polar or even charged side chains (positions 57 and 93 in all species, plus positions 116 in the horse and 140 in the human and the horse), while corresponding intraclass variation is small. All these structural features correlate with functional characteristics and suggest that the enzyme classes serve different roles. In addition, the replacements between these triad sets illustrate further general properties of the two mammalian alcohol dehydrogenase classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Model for the structure of formaldehyde dehydrogenase based on alcohol dehydrogenase

The three-dimensional structure of rat liver formaldehyde dehydrogenase (FALDH), previously known as class III alcohol dehydrogenase, was constructed using computer graphics and computer programs developed for model building. The construction is based on horse liver alcohol dehydrogenase (EE-ADH), whose structure has been elucidated by X-ray crystallography. The high sequence homology between the two enzymes makes knowledge-based modelling feasible in this case. The model shows a remarkable similarity to horse liver alcohol dehydrogenase especially in the NAD-binding domain. Certain mutations, and the one insertion in FALDH compared to EE-ADH in particular, have cause important changes in the substrate binding site, and thus aliphatic alcohols have been replaced by hemi-thioacetals as favourable substrates.     

Extensive variations and basic features in the alcohol dehydrogenase-sorbitol dehydrogenase family

Structural comparisons of sorbitol dehydrogenase with zinc-containing 'long' alcohol dehydrogenases reveal distant but clear relationships. An alignment suggests 93 positional identities with horse liver alcohol dehydrogenase (25% of 374 positions) and 73 identities with yeast alcohol dehydrogenase (20%). Sorbitol dehydrogenase forms a link between these distantly related alcohol dehydrogenases and is in some regions more similar to one of them that they are to each other. 43 residues (11%) are common to all three enzymes and include a heavy over-representation of glycine (half of all glycine residues in sorbitol dehydrogenase), showing the importance of space restrictions in protein structures. Four regions are well conserved, two in each domain of horse liver alcohol dehydrogenase. They are two segments close to the active-site zinc atom of the catalytic domain, and two in the central beta-pleated sheet strands of the coenzyme-binding domain. These similarities demonstrate the general importance of internal and central building units in proteins. Large variations affect a region adjacent to the third protein ligand to the active-site zinc atom in horse liver alcohol dehydrogenase. Such changes at active sites of related enzymes are unusual. Other large differences concern the segment around the non-catalytic zinc atom of horse liver alcohol dehydrogenase; three of its four cysteine ligands are absent from sorbitol dehydrogenase. Three segments with several exchanges correspond to a continuous region with superficial areas, inter-domain contacts and inter-subunit interactions in the catalytic domain of alcohol dehydrogenase. They may correlate with the altered quaternary structure of sorbitol dehydrogenase. Regions corresponding to top and bottom beta-strands in the coenzyme-binding domain of the alcohol dehydrogenase are also little conserved. Within sorbitol dehydrogenase, a large segment shows an internal similarity. The two distantly related alcohol dehydrogenases and sorbitol dehydrogenase form a triplet of enzymes illustrating basic protein relationships. They are ancestrally close enough to establish similarities, yet sufficiently divergent to illustrate changes in all but fundamental properties.     

Properties of 3-aldoxime pyridine adenine dinucleotide, an NAD analogue

The NAD⁺ analogue, 3-aldoxime pyridine adenine dinucleotide, is prepared by transglycosidation. Contrary to the published data, this analogue shows no activity as coenzyme with alcohol dehydrogenase from horse liver or from yeast. This is demonstrated by three methods: no increase of absorption at 331 nm by the enzymic oxidation of ethanol; no increase at 290 nm with cinnamic alcohol; and no exchange reaction. The inhibition by this analogue of the oxidation of ethanol by NAD⁺ is competitive at pH 7.6 and 9.5 with yeast alcohol dehydrogenase; with liver alcohol dehydrogenase, it is of the mixed type at pH 7.6 and non-competitive at pH 9.5. The lack of activity of the analogue and inhibition of the competitive or mixed type may be explained by the fact that the binary complex does not bind the substrate or that in the ternary complex the hydride shift does not occur. The non-competitive inhibition at pH 9.5 with the horse liver alcohol dehydrogenase may be explained by the existence of binding sites specific for this analogue.     

Interaction of yeast alcohol dehydrogenase with protoberberine alkaloids

Oxidation of ethanol and reduction of aldehyde catalysed by yeast alcohol dehydrogenase is inhibited by several naturally occurring as well as semi-synthetic protoberberine alkaloids. The affinity of these compounds for the enzyme depends essentially on their hydrophobicity. Corysamine and coptisine are the most potent inhibitors among the natural alkaloids of this group. The kinetics of yeast alcohol dehydrogenase inhibition with coptisine were analysed and equilibrium measurements using optical methods were carried out. The results suggest that the binding site of the enzyme for protoberberines is not identical with those for coenzyme and substrate though it should be located near the nicotinamide ring of bound NAD. The binding of protoberberines seems to be limited to rather superficially located hydrophobic groups in the vicinity of the active site of the enzyme. The inability of these alkaloids to protrude deeply into the molecule of yeast alcohol dehydrogenase at the catalytically important region is the main difference in their behaviour towards alcohol dehydrogenases from yeast and horse liver.     

Long-chained 1-mercapto-n-alkanes as potent inhibitors toward liver alcohol dehydrogenase

Long-chained 1-mercapto-n-alkanes showed potent inhibitory effects on horse liver alcohol dehydrogenase (HLADH). The inhibitory effect of the thiols was enhanced by increasing the number of the alkyl carbon atoms up to 10-12 and steeply lowered by further increase in the carbon number. The HLADH activity was almost completely inhibited in competitive manner by an equivalent concentration of 1-mercapto-n-decane or -n-dodecane to that of the subunit of the dimeric zinc enzyme; inhibition constant Ki was 0.55 nM for the former. The present study strongly suggests that the thiols interact simultaneously with at least two sites of HLADH; the primary one could be the zinc atom in the active site of the enzyme, interacting with the sulfhydryl groups, and the other a hydrophobic binding site for the their alkyl carbons.     

The use of biochemical solid-phase techniques in the study of alcohol dehydrogenase. 1. Some studies on subunit interactions in alcohol dehydrogenase with subunits covalently bound to agarose

The EE and SS isozymes of horse liver alcohol dehydrogenase have been immobilized separately to weakly CNBr-activated Sepharose 4B. The resulting immobilized dimeric preparations lost practically all of their activity after treatment with 6 M urea. However, enzyme activity was regenerated by allowing the urea-treated Sepharose-bound alcohol dehydrogenase to interact specifically with either soluble subunits of dissociated horse liver alcohol dehydrogenase or soluble dimeric enzyme. The regeneration of steroid activity in the immobilized preparations after treatment of the bound S subunits with soluble E subunits seems to show that true reassociation of the enzyme had taken place on the solid phase, since only isozymes with an S-polypeptide chain are active when using 5 beta-dihydrotestosterone as substrate. The results presented in this paper indicate that immobilized single subunits of horse liver alcohol dehydrogenase are inactive and that dimer formation is a prerequisite for the enzymic activity.