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

The binding of the coenzymes NAD+ and NADH to lactate dehydrogenase causes significant changes in the Raman spectra of both of these molecules relative to spectra obtained in the absence of enzyme. The molecular motions of the bound adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. We also have observed that the molecular motions of the bound adenine moiety are different from both those obtained when it is in either water, various hydrophobic solvents, or various other solvent compositions. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of NAD+ and the rocking motion of the carboxamide NH2 group of NADH. These shifts are probably caused by hydrogen bonding with the enzyme. The interaction energies of these hydrogen-bonding patterns are discussed. The aromatic nature of the nicotinamide moiety of NAD+ appears to be unchanged upon binding. Pronounced changes in the Raman spectrum of the nicotinamide moiety of NADH are observed upon binding; some of these changes are understood and discussed. Finally, these results are compared to analogous results that were recently reported for liver alcohol dehydrogenase [Chen et al. (1987) Biochemistry 26, 4776-4784]. In general, the coenzyme binding properties are found to be quite similar, but not identical, for the two enzymes.     

Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques

We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Raman study of reduced nicotinamide adenine dinucleotide bound to liver alcohol dehydrogenase

We report the first Raman spectra of reduced nicotinamide adenine dinucleotide (NADH) when bound to an enzymatic active site, that of liver alcohol dehydrogenase (LADH). This was obtained by subtracting the Raman spectrum of LADH from that of the binary LADH/NADH complex. There are significant changes in the spectrum of bound NADH as compared to that in solution. The data indicate that both the nicotinamide moiety and the adenine moiety are involved in the binding. At least one of the two NH2 moieties of NADH also participates.     

Electron transfer from NADH bound to horse liver alcohol dehydrogenase (NAD+ dependent dehydrogenase): visualisation of the activity in the enzyme crystals and adsorption of formazan derivatives by these crystals

The crystals of holoenzyme from native and cross-linked alcohol dehydrogenase exhibit electron transfer from NADH to phenazinium methosulfate (PMS), and then to the tetrazolium salt sodium 3,3'-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonate (XXT). The slow dissociation of the cofactor and/or the conformational change associated can now be bypassed. The reduction product, formazan, did not diffuse out of the crystals in buffer and the crystals turned colored. In the presence of dimethyl sulfoxide or dimethoxyethane, the formazan diffused out to the solution. The reaction rates were found to be, respectively, 18% and 15% of the redox reaction rate of ethanol with cinnamaldehyde, close to the activity determined for the enzyme in solution in the presence of dimethoxyethane. The use of system PMS-tetrazolium salt is a useful tool to visualize the activity of dehydrogenases and other electron transferring systems in the crystalline state. The adsorption of formazan by the alcohol dehydrogenase crystals occurs in solution.     

Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase

Time-resolved fluorescence studies on the emission of NADH bound to porcine heart mitochondrial malate dehydrogenase [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37), in the presence and absence of saturating levels of hydroxymalonate, were carried out. The lifetime of NADH bound in the ternary complex was determined to be 9.5 ns compared to 1.74 ns as reported in the literature. Steady-state and dynamic polarization data indicated a Debye rotational relaxation time in the range of 106-109 ns for the dimeric enzyme. This value is significantly larger than that calculated for a spherical protein and is consistent with the asymmetric dimer found by crystallographic studies.     

Multiple conformations of NAD and NADH when bound to human cytosolic and mitochondrial aldehyde dehydrogenase

Crystallographic analysis revealed that the nicotinamide ring of NAD can bind with multiconformations to aldehyde dehydrogenase (ALDH) (Ni, L., Zhou, J., Hurley, T. D., and Weiner, H. (1999) Protein Sci. 8, 2784-2790). Electron densities can be defined for two conformations, neither of which appears to be compatible with the catalytic reaction. In one conformation, it would prevent glutamate 268 from functioning as a general base needed to activate the catalytic nucleophile, cysteine 302. In the other conformation, the nicotinamide is too far from the enzyme-substrate adduct for efficient hydride transfer. In this study, NMR and fluorescence spectroscopies were used to demonstrate that NAD and NADH bind to human liver cytosol and mitochondrial ALDH such that the nicotinamide samples a population of conformations while the adenosine region remains relatively immobile. Although the nicotinamide possesses extensive conformational heterogeneity, the catalyzed reaction leads to the stereospecific transfer of hydride to the coenzyme. Mobility allows the nicotinamide to move into position to be reduced by the enzyme-substrate adduct. Although the reduced nicotinamide ring retains mobility after NADH formation, the extent of the motion is less than that of NAD. It appears that after reduction the population of favored nicotinamide conformations shifts toward those that do not interfere with the ability of the enzyme to release the reaction product. In the case of the mitochondrial, but not the cytosolic, enzyme this change in conformational preference is promoted by the presence of Mg2+ ions. Coenzyme conformational mobility appears to be beneficial to catalysis by ALDH throughout the catalytic cycle.     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

Raman spectroscopy of liver alcohol dehydrogenase

We report the Raman spectrum of liver alcohol dehydrogenase in solution. The enzyme's secondary structure as determined from an examination of the Raman bands is slightly different than that found in crystals by X-ray diffraction.     

Conformation of NAD+ bound to yeast and horse liver alcohol dehydrogenase in solution. The use of the proton-proton transferred nuclear Overhauser enhancement

Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B-form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G₁ cycling versus G₀ non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.     

NAD+ and NAD+ analogues in horse liver alcohol dehydrogenase. Relationship between reactivity and conformation simulated with molecular mechanics

In the present study we show that the enzymatic activity of the coenzyme nicotinamide adenine dinucleotide (NAD+) and its analogues (C(O)NH2 replaced by C(S)NH2, C(O)CH3, C(O)H and CN) with horse liver alcohol dehydrogenase (LADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) can be rationalized by their conformation in the active site determined with molecular mechanics (AMBER, assisted model building with energy refinement). In order to establish the relation between the hydride transfer rate and the conformation of the NAD+ and its analogues, kinetic experiments with the poor substrate isopropanol were carried out. It appears that the enzymatic activity can be readily explained by the geometry of the pyridinium ring, in particular the magnitude of the 'out-of-plane' rotation of the carboxamide side chain (or analogues). The latter is nicely illustrated in the case of 3-cyanopyridine adenine dinucleotide which lacks any 'out-of-plane' rotation and concomitantly exhibits no significant enzymatic activity.     

Molecular dynamics simulations of NAD+-induced domain closure in horse liver alcohol dehydrogenase

Horse liver alcohol dehydrogenase is a homodimer, the protomer having a coenzyme-binding domain and a catalytic domain. Using all available x-ray structures and 50 ns of molecular dynamics simulations, we investigated the mechanism of NAD+-induced domain closure. When the well-known loop at the domain interface was modeled to its conformation in the closed structure, the NAD+-induced domain closure from the open structure could be simulated with remarkable accuracy. Native interactions in the closed structure between Arg369, Arg47, His51, Ala317, Phe319, and NAD+ were seen to form at different stages during domain closure. Removal of the Arg369 side-chain charge resulted in the loss of the tendency to close, verifying that specific interactions do help drive the domains closed. Further simulations and a careful analysis of x-ray structures suggest that the loop prevents domain closure in the absence of NAD+, and a cooperative mechanism operates between the subunits for domain closure. This cooperative mechanism explains the role of the loop as a block to closure because in the absence of NAD+ it would prevent the occurrence of an unliganded closed subunit when the other subunit closes on NAD+. Simulations that started with one subunit open and one closed supported this.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase.

The monophoton counting technique was used to obtain the fluorescence decay kinetics of NADH (dihydronicotinamide adenine dinucleotide) bound to LADH (HORSE LIVER ALCOHOL DEHYDROGENAS). It was found that the fluorescence decay of the enzyme complex did not follow a single exponential decay law but that the data could be well described as a sum of two exponentials. The decay parameters of the enzyme complex do not depend on the degree of binding-site saturation. These results are interpreted in terms of a reversible excited-state reaction forming a nonfluorescent product. Fluorescence decay kinetics are also reported for NADH and related molecules in solution. The decay parameters, fluorescence emission maxima, and fluorescence intensities depend on solvent polarity and viscosity.