Characterization of a transient intermediate formed in the liver alcohol dehydrogenase catalyzed reduction of 3-hydroxy-4-nitrobenzaldehyde

The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pKa = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pKa = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations (lambda f approximately equal to 3 lambda s). Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and 2H kinetic isotope effects. It was found that NADH oxidation takes place at the rate of the slower relaxation (lambda s); when NADD is substituted for NADH, lambda s is subject to a small primary isotope effect (lambda Hs/lambda Ds = 2.0); and the events that occur in lambda s precede lambda f. These findings identify the intermediate as a ternary complex containing bound oxidized nicotinamide adenine dinucleotide (NAD+) and some form of 3-hydroxy-4-nitrobenzyl alcohol. The blue-shifted spectrum of the intermediate strongly implies a structure wherein the phenolic hydroxyl is neutral. When constrained to a mechanism that assumes only the neutral phenolic form of the substrate binds and reacts and that the intermediate is an E(NAD+, product) complex, computer simulations yield RSSF and single-wavelength time courses that are qualitatively and semiquantitatively consistent with the experimental data. We conclude that the LADH substrate site can be divided into two subsites: a highly polar, electropositive subsite in the vicinity of the active-site zinc and, just a few angstroms away, a rather nonpolar region. The polar subsite promotes formation of the two interconverting reactive ternary complexes. The nonpolar region is the binding site for the hydrocarbon-like side chains of substrates and in the case of 3-hydroxy-4-nitrobenzaldehyde conveys specificity for the neutral form of the phenolic group.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of 4-trans-(N,N-dimethylamino)cinnamaldehyde with the liver alcohol dehydrogenase-oxidized nicotinamide adenine dinucleotide complex

Evidence that horse liver alcohol dehydrogenase forms a ternary complex with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) and oxidized nicotinamide adenine dinucleotide (NAD+) is presented. Formation of the complex is characterized by a 97-nm red shift of the free chromophore to 495 nm (epsilon 495 approximately 6.0 X 10(4) M-1 cm-1). This shift is larger than the 66-nm red shift of the E(NADH,-DACA) complex (lambda max = 464 nm) previously reported by Dunn and Hutchinson [Dunn, M.F., & Hutchison, J.S. (1973) Biochemistry 12, 4882-4892]. The large red shift of the E(NAD+,DACA) complex is due to the combined effects of coordination of the carbonyl oxygen of DACA to the active-site zinc ion and to the close proximity of the positively charged nicotinamide ring of NAD+. The stability of this complex is pH dependent and depends on a single apparent ionization with pKa = 7.6 +/- 0.3. The pH-independent dissociation constant for binding of DACA to E(NAD+) is 23 +/- 6 microM. The stoichiometry of DACA binding to the E(NAD+) complex is shown to be one per active site (two per enzyme molecule). Liver alcohol dehydrogenase is also shown to catalyze the NAD+-mediated oxidation of DACA to the corresponding carboxylic acid with a very slow turnover rate. The possibility that the observed E(NAD+,DACA) complex is an intermediate in the enzyme-catalyzed oxidation of DACA is discussed.     

Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

19F nuclear magnetic resonance observations of aldehyde dismutation catalyzed by horse liver alcohol dehydrogenase

We have examined aspects of the second catalytic activity of alcohol dehydrogenase from horse liver (LADH), which involves an apparent dismutation of an aldehyde substrate into alcohol and acid in the presence of LADH and NAD. Using the substrate p-trifluoromethylbenzaldehyde, we have observed various bound complexes by ¹⁹F NMR in an effort to further characterize the mechanism of the reaction. The mechanism appears to involve the catalytic activity of LADH · NAD · aldehyde complex which reacts to form an enzyme · NADH · acid complex. The affinity of the acid product for LADH · NADH is weak and the acid product readily desorbs from the ternary complex. The resulting LADH · NADH can then react with a second molecule of aldehyde to form NAD and the corresponding alcohol. The result is the conversion of two molecules of aldehyde to one each of acid and alcohol, with LADH and NAD acting catalytically. This sequence of reactions can also explain the slow formation of acid product observed when alcohol and NAD are incubated with the enzyme.     

Novel substrates of yeast alcohol dehydrogenase--4. Allyl alcohol and ethylene glycol

In the present work, we have determined the steady-state kinetic constants for yeast alcohol dehydrogenase-catalyzed oxidation of allyl alcohol (H2C = CH.CH2OH) and ethylene glycol (HOCH2.CH2OH) with NAD+, at pH 8.0; also, a kinetic mechanism for the former reaction was determined at the same pH. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol dehydrogenase.     

Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.     

Molecular properties of p-(dimethylamino)benzaldehyde bound to liver alcohol dehydrogenase: a Raman spectroscopic study

We have studied the binding nature of an aromatic aldehyde to the catalytic site of liver alcohol dehydrogenase from horse (LADH) using preresonance Raman spectroscopy. The compound p-(dimethylamino)benzaldehyde (DABA) is converted to the corresponding alcohol in the presence of nicotinamide adenine dinucleotide (NADH) and a catalytic amount of enzyme at neutral pH. A stable ternary complex of LADH/NADH/DABA can be formed if enzyme and coenzyme are in excess at high pH [Jagodzinski, P. W., Funk, G. F., & Peticolas, W. L. (1982) Biochemistry 21, 2193-2202]. We have obtained the preresonance Raman spectrum of bound DABA by subtracting the contribution of the binary complex of LADH/NADH from the spectrum of this stable ternary complex. In order to understand the normal mode patterns of DABA, four isotopically labeled DABA derivatives were synthesized and their Raman spectra, in solution and in the ternary complex, were measured. Three of these compounds contain substitutions in the functionally important aldehyde moiety: (i) In one such substitution, the aldehydic hydrogen atom was replaced by a deuterium; (ii) in another, this hydrogen atom was replaced by deuterium, and the aldehydic carbon atom was replaced by 13C; and (iii) in the third derivative, only the carbon atom was replaced by 13C. The fourth derivative has had the two hydrogen atoms at the 3- and 5-positions of the DABA ring replaced by deuterium atoms. We find that many of the spectral modes are fairly extended, involving both stretching and bending motions of the entire molecule, although a few modes are quite localized. We find that the normal mode structure of DABA changes considerably when it binds to LADH/NADH. As a model for the bound DABA, we have examined the zinc complexes of DABA (and all four isotopically labeled samples) in anhydrous diethyl ether and methylene chloride. A striking correspondence between the Raman spectra of the enzyme-bound DABA and DABA-Zn complexes in solution is found, which extends to all the isotopically labeled derivatives. This suggests that one of the major roles of LADH in the binding of DABA is to provide a divalent zinc ion to form a first-sphere Lewis acid complex. The data also suggest other interactions between enzyme-bound DABA with its protein surroundings and with the coenzyme NADH are quite minor. An estimate of the carbonyl bond character of bound DABA had been made on the basis of the response of Raman bands to isotopic labeling and on trends observed in spectra of DABA in solvents of various polarities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal stability of nicotinamide adenine dinucleotide dependent dehydrogenases.

The technique of differential scanning calorimetry (DSC) has been applied to the study of temperature-induced irreversible denturation and thus to the heat stability of soluble and Sepharose-bound liver alcohol dehydrogenase (LADH, EC 1.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) in the presence of various coenzymes or coenzyme fragments. The transition temperature (Ttr) of 82.5 degrees C obtained for soluble LADH was increased by 12.5 degrees C in the presence of a saturating concentration of NACH. In the presence of NAD+, Ttr increased by 8.5 degrees C, whereas ADP-ribose and AMP caused an increase in Ttr of only 2 and 1 degree C, respectively. The Ttr of 85.5 degrees C obtained for Sepharose-bound LADH was increased by about 12 degrees C after the addition of free NADH. However, when the enzyme was immobilized simultaneously with a NADH analogue (which also binds to the matrix), a broad endotherm with a Ttr of 91.5 degrees C was obtained, indicating the presence of immobilized enzyme molecules both with, and without, associated NADH. Corresponding increases in heat stability were observed for LDH in solution in the presence of NADH, NAD+, and AMP, leading to increases in Ttr from 72 to 79.5 and 74 and 73 degrees C, respectively. The addition of pyruvate and NAD+ to the enzyme to form an abortive ternary complex led to the same stabilization as that observed with NADH, attendant with a large increase in the enthalpy of transition, deltaHtr. In these studies the technique of DSC was utilized because it is applicable both to soluble and immobilized enzymes and (1) provides rapid information about Ttr and thus thermal stability of enzymes, (2) different energetic states of an enzyme molecule can be identified, and (3) an overall picture of the thermal process is rapidly obtained.     

Carboxymethylated liver alcohol dehydrogenase: kinetic and thermodynamic characterization of reactions with substrates and inhibitors

Liver alcohol dehydrogenase (LADH) carboxymethylated at Cys-46 (CMLADH) forms two different ternary complexes with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA). The complex with reduced nicotinamide adenine dinucleotide (NADH) is characterized by a 38-nm red shift of the long-wavelength pi, pi* transition to 436 nm, while the complex with oxidized nicotinamide adenine dinucleotide (NAD+) is characterized by a 60-nm red shift to 458 nm. CMLADH also forms a ternary complex with NAD+ and the Z isomer of 4-trans-(N,N-dimethylamino)cinnamaldoxime in which the absorption of the oxime (lambda max = 354 nm) is red shifted 80 nm to 434 nm. Pyrazole and 4-methylpyrazole are weak competitive inhibitors of ligand binding to the substrate site of native LADH. These inhibitors were found to form ternary complexes with CMLADH and NADH which are more stable than the corresponding complexes with the native enzyme. The transient reductions of the aldehydes DACA and p-nitrobenzaldehyde (NBZA) were studied under single-turnover conditions. Carboxymethylation decreases the DACA reduction rate 80-fold and renders the process essentially independent of pH over the region 5-9, whereas this process depends on a pKa of 6.0 in the native enzyme. At pH 7.0, the rate constant for NBZA reduction also is decreased at least 80-fold to a value of 7.7 +/- 0.3 s-1. Since primary kinetic isotope effects are observed when NADH is substituted with (4R)-4-deuterio-NADH (kH/kD = 3.0 for DACA and kH/kD = 2.3 for NBZA), the rate-limiting step for both aldehydes involves hydride transfer. The altered pH dependence is concluded to be due to an increase in the pK value of the zinc-coordinated DACA-alcohol in the ternary complex with NAD+ by more than 3 units. This perturbation is brought about by the close proximity of the negatively charged carboxymethyl carboxylate.     

Coordination geometry for cadmium in the catalytic zinc site of horse liver alcohol dehydrogenase: studies by PAC spectroscopy

Active site substituted Cd(II) horse liver alcohol dehydrogenase has been studied by Perturbed Angular Correlation of Gamma rays Spectroscopy during turnover conditions for benzaldehyde and 4-trans-(N,N-dimethylamino)cinnamaldehyde. The ternary complex between alcohol dehydrogenase NAD⁺ and Cl^–, and the binary complex between alcohol dehydrogenase and orthophenanthroline have also been studied. The Nuclear Quadrupole Interaction parameters have been interpreted in terms of different coordination geometries for Cd(II) in the catalytic zinc site of the enzyme. Calculation of the nuclear quadrupole interaction for cadmium in the catalytic site of the enzyme with and without coenzyme, based upon the four coordinated geometries determined from X-ray diffraction, agrees with the experimentally determined values. The ternary complexes between enzyme, NAD⁺ and either Cl^– or trifluoroethanol and the binary complex between enzyme and orthophenanthroline have almost identical spectral parameters which are not consistent with a four coordinated geometry, but are consistent with a five coordinated geometry. The nonprotein ligands for the ternary complex with trifluoroethanol are suggested to be an alkoxide group and a water molecule. The Nuclear Quadrupole Interaction parameters for the productive ternary complex between enzyme, NADH and an aldehyde is consistent with the four coordinated geometry predicted from X-ray diffraction data having the carbonyl group of the aldehyde substituting the water molecule as ligand to the metal.Abbreviations LADH Horse liver alcohol dehydrogenase - H₄Zn₂LADH derivative of LADH free of zinc in the catalytic site - ¹¹¹CdZn₂LADH derivative of LADH with ¹¹¹Cd (carrier free) in the catalytic site - Cd₂Zn₂LADH derivative of LDH with 2 mole of Cd(II) per mole LADH in the catalytic site - PAC pertubed angular correlation of gamma rays - NQI Nuclear quadrupole interaction - AOM Angular overlap model - trifluoroethanol 2,2,2-trifluoroethanol - DACA trans-4-(N,N-dimethylamino)cinnamaldehyde - NAD⁺ and NADH oxidized and reduced nicotinamide adenine dinucleotide - NADH₂ reduced 1,4,5,6-tetrahydronicotinamide adenine dinucleotide The experimental work was carried out at the Niels Bohr Institute Risø, 4000 Roskilde and Blegdamsvej 19, 2100 Copenhagen, Denmark Offprint requests to: R. Bauer     

Secondary 15N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases

Secondary 15N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these 15N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer [Cook, P. F., Oppenheimer, N. J. & Cleland, W. W. (1981) Biochemistry 20, 1817]. If this mechanism were valid, as N-1 becomes pyramidal an 15N isotope effect of up to 2-3% would be observed. In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant 15N kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.     

Spectral evidence for three metal-linked ionization equilibria in the interaction of cobalt(II) horse liver alcohol dehydrogenase with coenzyme and substrate

The visible absorption bands in the region 525-575 nm of the catalytic cobalt ion in cobalt(II) horse liver alcohol dehydrogenase show characteristic pH-dependent changes both in the free enzyme and its complexes with nicotinamide adenine dinucleotide (NAD+) and NAD+ plus ethanol or 2,2,2-trifluoroethanol. In the free enzyme, the change of the coordination environment has an apparent pK of about 9.4. In the binary complex with NAD+ the spectral changes are complex, indicating changes in the coordination sphere in a lower pH range with an estimated pK value of about 7.9. The ternary complexes enzyme X NAD+ X ethanol and enzyme X NAD+ X 2,2,2-trifluoroethanol exhibit very similar, characteristic spectral features; their apparent pK values are 6.3 and less than 4, respectively. We ascribe these pK values to the ionization of the alcohol bound in the ternary complexes. The results demonstrate that the catalytic cobalt ion is sensing changes of the ionization state of the protein when going from low pH forms to high pH forms both in the absence and presence of coenzyme and substrate/inhibitor.     

Probes of a role for remote binding interactions on hydrogen tunneling in the horse liver alcohol dehydrogenase reaction

A tunneling contribution to hydride transfer has been demonstrated previously in the oxidation of benzyl alcohol catalyzed by an active-site mutant (F93W) of horse liver alcohol dehydrogenase (LADH) [Bahnson, B. J., et al. (1993) Biochemistry 32, 5503-5507]. Mutation of a residue that lies directly behind the nicotinamide ring of the bound cofactor has further shown that side-chain bulk can contribute to catalytic efficiency and tunneling in a correlated fashion [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. Second site mutations of F93W have now been made at positions more remote from the active site. In particular, we have focused on an isoleucine residue that interacts with the adenine moiety of the NAD(+) cofactor, 20 A from the nicotinamide ring. Replacement of this remote residue with glycine (F93W:I224G), alanine (F93W:I224A), valine (F93W:I224V), and leucine (F93W:I224L) is concluded to destabilize the binding of NAD(+). All double mutants exhibited a K(M) for NAD(+) that is 2-25 times higher than that for the F93W enzyme. However, neither the catalytic efficiency for turnover of benzyl alcohol [k(cat)/K(M(benzyl alcohol))] nor the relationship between the secondary k(H)/k(T) and k(D)/k(T) isotope effects for benzyl alcohol oxidation was significantly affected. The lack of differences observed in the isotope effects indicates that these mutations have little effect on the extent of hydrogen tunneling in the reaction. The complete removal of the side chain at position 224 in the F93W:I224G enzyme resulted in a less than 5% decrease in the ratio of the secondary isotope effects, maintaining the ratio above the semiclassical limit for the indication of tunneling in the reaction. By contrast, K(i) for NAD(+) increased 60-fold for this mutant. The results obtained with F93W:I224G are consistent with remote interactions that affect the association and binding of cofactor in a reactive conformation. However, once this conformation is achieved, hydride transfer and its tunneling component proceed as with the single F93W mutant enzyme, uninfluenced by the remote mutation. Replacement of other side chains, with alpha-carbon positions from about 8 to over 20 A from the C4 position of the nicotinamide ring, demonstrated a similar insensitivity of k(cat)/K(M(benzyl alcohol)) to protein modification. Comparison to earlier studies with active-site mutants of LADH implicates a role for proximal, but not distal, side chains in the modulation of hydrogen tunneling for this enzyme.     

pH-dependent conformational states of horse liver alcohol dehydrogenase.

The quenching of liver alcohol dehydrogenase protein fluorescence at alkaline pH indicates two conformational states of the enzyme with a pKa of 9.8+/-0.2, shifted to 10.6+/-0.2 in D2O. NAD+ and 2-p-toluidinonaphthalene-6-sulfonate, a fluorescent probe competitive with coenzyme, bind to the acid conformation of the enzyme. The pKa of the protein-fluorescence quenching curve is shifted toward 7.6 in the presence of NAD+, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench. At pH (pD) 10.5, the rate constant for NAD+ binding was 2.6 times faster in D2O2 than in H2O due to the shift of the pKa. Based on these results, a scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme. NAD+ binds to the acid conformation and subsequently causes another conformational change resulting in the perturbation of the pKa to 7.6. Alcohol then binds to the unprotonated form of the functional group with a pKa of 7.6 in the binary enzyme-NAD+ complex and converts the enzyme to the alkaline conformation. Thus, at neutral pH liver alcohol dehydrogenase undergoes two conformational changes en route to the ternary complex in which hydride transfer occurs.     

Fluorescence quenching of Trp-314 of liver alcohol dehydrogenase by oxygen

The quenching of the fluorescence of liver alcohol dehydrogenase (LADH) by molecular oxygen has been studied by both fluorescence lifetime and intensity measurements. This was done in the presence of 1 M acrylamide which selectively quenches the fluorescence of the surface tryptophan residue, Trp-15, thus allowing us to focus on the quenching of the deeply buried tryptophan, Trp-314, by molecular oxygen. Such studies yielded a Stern-Volmer plot of F0/F with a greater slope than the corresponding tau o/tau plot. This indicates that both dynamic and static quenching of Trp-314 occurs. The temperature dependence of the dynamic quenching of LADH by oxygen was also studied at three temperatures, from which we determined the activation enthalpy for the quenching of Trp-314 to be about 10 kcal/mol. The oxygen quenching of a ternary complex of LADH, NAD+ and trifluoroethanol was also studied. The rate constant for dynamic quenching of Trp-314 by oxygen was found to be approximately the same in the ternary complex as that in the unliganded enzyme.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Effect of substrates and coenzymes on the molecular symmetry-related properties of horse liver and yeast alcohol dehydrogenases

Thermal inactivation of horse liver alcohol dehydrogenase (LADH) exhibits the following biphasic kinetics A = Afast.e-Kfast.t + Aslow.e-Kslow.t Where A is the per cent residual activity at time t,Afast and Aslow are amplitudes (expressed as % of initial activity) and kfast and kslow first-order rate constants of the fast and slow phases, respectively. For apoenzyme, Afast = Aslow = 50% of the initial activity under all conditions of temperature and pH. On the addition of a substrate or coenzyme ligand, there is a ligand concentration-dependent increase in per cent Aslow and a decrease in kslow. At sufficiently high ligand concentration, the entire time-course of inactivation can be described as a single exponential decay. The variations of per cent Aslow and of kslow with ligand concentration are consistent with the existence of two binding sites of different ligand affinities. Inactivation of LADH by excess EDTA also exhibits a similar biphasic kinetics with Afast = Aslow = 50% of the initial activity. Addition of ethanol or NAD+ brings about a concentration-dependent decrease in kfast and kslow without affecting amplitudes of the two phases. The NAD+ concentration-dependence of this decrease is consistent with a single dissociation constant for the coenzyme. Inactivation of yeast alcohol dehydrogenase by heat or excess EDTA can be represented as a single exponential decay of activity under all conditions of temperature and pH in the absence as well as presence of ethanol or NAD+. Implications of these results for the molecular symmetry of the two oligomeric enzymes in solution are discussed.