Lifetimes and NADH quenching of tryptophan fluorescence in pig heart cytoplasmic malate dehydrogenase.

The time-resolved and steady state fluorescence properties were measured for pig heart cytoplasmic malate dehydrogenase at pH 6.0 and 8.0. The fluorescence decay can be described by two rate processes, according to the functions: I(t) = 0.7e(-t/1.0) + 0.3e(-t/4.4) for the free enzyme and I(t) = 0.7e(-t/0.8) + 0.3e(-t/2.0 for the enzyme . NADH complex. Quenching by NADH of the tryptophan fluorescence is linear. The only effect of pH is to change the association constant for NADH binding; the fluorescence of the free enzyme and the fluorescence quenching by NADH, I-, and acrylamide are unaffected by pH. Thus there are no changes in conformation of the free enzyme or of the NADH complex over the range of pH 6 to 8.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

Pulse fluorimetry study of beef liver glutamate dehydrogenase reduced nicotinamide adenine dinucleotide phosphate complexes.

Single photon counting pulse fluorimetry has been used in order to study the two ternary complexes GDH-GTP-NADPH and GDH-L-glutamate-NADPH and the quaternary complex GDH-GTP-L-glutamate-NADPH. The fluorescence decay of the enzyme-bound NADPH is not monoexponential in any of these complexes. Moreover, it does not seem to be dependent on the coenzyme concentration. The experimental curves can be satisfactorily fitted with the sum of two exponentials, the relative amplitudes of which significantly depend on the complex studied. Thus, for dihydronicotinamide two possible environments might exist in the enzyme active sites. It is also shown that the fluorescence decay times of the enzyme are shortened by the bound NADPH.     

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.     

Characterization of the mechanism of the NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4: Formation of a productive NADH-enzyme complex and its role in the general mechanism of NADH and FAD-dependent enzymes

The NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4 is a member of the single cysteine-containing subset of the family of disulfide reductases represented by glutathione reductase. We have determined the kinetics of the reductive half-reaction of the enzyme with NADH using stopped-flow spectroscopy and kinetic isotope effects, and these results indicate that the reductive and oxidative half-reactions are both partially rate-limiting for enzyme turnover. During reaction with NADH, the reduced nucleotide appears to bind rapidly in an unproductive conformation, followed by the formation of a productive E·NADH complex and subsequent electron transfer to FAD. F161 of Npsr fills the space in which the nicotinamide ring of NADH would be expected to bind. We have shown that while this residue is not absolutely required for catalysis, it does assist in the forward commitment to catalysis through its role in the reductive half reaction, where it appears to enhance hydride transfer in the productive E·NADH complex. While the fluorescence and absorbance spectra of the stable redox forms of the wild-type and F161A mutant enzymes are similar, intermediates formed during reduction and turnover have different characteristics and appear to indicate that the enzyme–NADH complex formed just prior to hydride transfer on the F161A enzyme has weaker FAD–NADH interactions than the wild-type enzyme, consistent with a “looser” enzyme–NADH complex. The 2.7 Å crystal structure of the F161A mutant was determined, and shows that the nicotinamide ring of NADH would have the expected freedom of motion in the more open NADH binding cavity.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of enzyme-ligand complexes using dynamic fluorescence anisotropy. II. The coenzyme-binding site of malate dehydrogenase

The coenzyme-binding site in mitochondrial malate dehydrogenase from pig heart was studied using dynamic fluorescence anisotropy decay. The dynamics of the fluorescent ligands NADH and 6-cyano-7-hydroxy-4,8-dimethylcoumarin were used to detect conformational changes at the dihydronicotinamide-and at the adenosine-binding sites, respectively. Addition of the natural substrate L-malate to the complex from enzyme and NADH does not influence the complete immobilization of the dihydronicotinamide group, whereas the stereoisomer D-malate and the substrate-analogue hydroxymalonate form ternary complexes with highly mobile dihydronicotinamide. The dynamics of the fluorescent adenosine-analogue are not influenced by formation of complexes with substrate and substrate-analogues. Thus the conformational changes at the dihydronicotinamide-binding site remain local and are not transmitted to the adenosine-binding site.     

Rapid relaxation processes in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens revealed by subnanosecond-resolved laser-induced fluorescence

Time-resolved fluorescence studies were carried out on the FAD bound to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. The transient fluorescence exhibits complex decay kinetics with at least a short lifetime component in the 50-500-ps time region and a longer one in the range 1.5-3.5 ns. The shorter-lifetime component has a larger contribution in the presence of substrate (p-hydroxybenzoate) or inhibitor (p-aminobenzoate). The quenching of the fluorescence is both static and dynamic in nature. The decay of fluorescence anisotropy shows that the FAD environment is both flexible and rigid. The FAD mobility can be enhanced by dilution of the enzyme, by raising the temperature, or by the binding of substrate or inhibitors. The anisotropy results are interpreted in part in terms of a monomer-dimer equilibrium, whereby the FAD in the monomer contains much more flexibility. The above-mentioned effects induce a shift of the equilibrium to the monomeric side. From a constrained parameter fitting the dissociation constant is estimated to be about 1 microM for the free enzyme and somewhat higher for the binary complexes between the enzyme and substrate or inhibitor. pH variation has only a slight effect on fluorescence or anisotropy decay parameters, while dimethylsulfoxide appears to promote dissociation into monomers by weakening hydrophobic interaction between the subunits. The results are discussed in the light of newly developed insights into the functional role of rapid structural fluctuations in enzyme catalysis.     

Differences between the reactivities of two pyridine nucleotides in the rapid reduction process and the reoxidation process of adrenodoxin reductase.

The reaction process of adrenodoxin reductase with NADPH and NADH were investigated. The appearance of new intermediate with a broad absorption band at around 520 nm has been detected by rapid-scan stopped-flow spectrophotometry. Although the formation of this intermediate is more rapid with NADPH than with NADH, the rates of the subsequent decay to the fully reduced state are almost identical (Kobs values were 20.5 and 16.0s-1). These results indicate that the new intermediate is the complex formed between the oxidized enzyme and reduced pyridine nucleotide (enzyme-substrate complex), and that subsequent decay of the intermidiate is caused by a two-electron transfer process from the reduced pyridine nucleotide to the enzyme flavin. On the other hand, spectral and kinetic properties in the steady state of the reoxidation reaction of the enzyme reduced with NADPH and NADH were somewhat different. The rate of reoxidation of the enzyme under aerobic conditions from the reduced state to the oxidized state was 6.5 times faster when a 10-fold molar excess of NADH was used than when NADPH of the same concentration was used. This result is consistent with the fact that the NADH-dependent oxidase activity was 6.4 times greater than that dependent on NADPH. During reoxidation of the reduced enzyme under aerobic conditions in the presence of an excess of NADPH or NADH, the EPR spectra indicated the formation of the flavin semiquinone radical species. Similarly, the formation of semiquinone was observed in the absorption spectrum with either NADPH or NADH under the same conditions as in the EPR measurement. The intensity of the semiquinone signal on EPR was considerably smaller with NADH than with NADPH. These results suggest that NADP+ complex with the enzyme semiquinone protects the radical from oxidation by oxygen to a greater extent than NAD+, and consequently the semiquinone is easier to detect with NADPH than with NADH.     

Light-induced fluorescence decay during the greening of normal and lincomycin-treated maize leaves

Light-induced fluorescence decay was examined during the greening of control and lincomycintreated maize (Zea mays L.) leaves. Assuming that this decay to a first approximation is the result of two parallel first-order reactions, the fluorescence induction curves were linearized on the logarithm plot and the parameters were determined. The variable fluorescence increased, and the parameters of the two linear sections of the fluorescence decay—that is, the kinetics of the induction curves—changed during the greening of the control leaves. Lincomycin treatment caused some chlorophyll deficiency and the lowering of the chlorophyll a/b ratio, changed the fluorescence emission spectra and the effect of Mg²⁺ on the regulation of the excitation energy distribution. The structure of the thylakoids and the kinetics of the fluorescence decay were also changed in the treated leaves. The possible relationship between the change of the kinetics of the fluorescence decay and the change of spillover during greening and after lincomycin treatment is discussed.Abbreviations LHC light-harvesting complex - Chl chlorophyll - LM lincomycin - PS photosystem - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from synechocystis

Chlorophyll fluorescence decay kinetics in photosynthesis are dependent on processes of excitation energy transfer, charge separation, and electron transfer in photosystem II (PSII). The interpretation of fluorescence decay kinetics and their accurate simulation by an appropriate kinetic model is highly dependent upon assumptions made concerning the homogeneity and activity of PSII preparations. While relatively simple kinetic models assuming sample heterogeneity have been used to model fluorescence decay in oxygen-evolving PSII core complexes, more complex models have been applied to the electron transport impaired but more highly purified D1-D2-cyt b(559) preparations. To gain more insight into the excited-state dynamics of PSII and to characterize the origins of multicomponent fluorescence decay, we modeled the emission kinetics of purified highly active His-tagged PSII core complexes with structure-based kinetic models. The fluorescence decay kinetics of PSII complexes contained a minimum of three exponential decay components at F(0) and four components at F(m). These kinetics were not described well with the single radical pair energy level model, and the introduction of either static disorder or a dynamic relaxation of the radical pair energy level was required to simulate the fluorescence decay adequately. An unreasonably low yield of charge stabilization and wide distribution of energy levels was required for the static disorder model, and we found the assumption of dynamic relaxation of the primary radical pair to be more suitable. Comparison modeling of the fluorescence decay kinetics from PSII core complexes and D1-D2-cyt b(559) reaction centers indicated that the rates of charge separation and relaxation of the radical pair are likely altered in isolated reaction centers.     

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy

NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor-fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm.     

Bull semen nicotinamide adenine dinucleotide nucleosidase. VI. Fluorescence studies of the enzyme with reduced nicotinamide adenine dinucleotide

The binding of NADH to bull semen NAD nucleosidase was observed to be accompanied by a considerable enhancement of the fluorescence of NADH. The fluorescence enhancement observed in the binding of NADH to the enzyme was utilized to study the stoichiometry of binding of this compound to the enzyme. Results obtained from the fluorescence titration of the enzyme with NADH indicated the binding of one mole of NADH per mole of enzyme (36,000 g). The dissociation constant for the enzyme-NADH complex was determined to be 2.52 × 10^?6m. NADH was also found to be a very effective competitive inhibitor of the NADase-catalyzed hydrolysis of NAD, and the inhibitor dissociation constant (K_I) for the enzyme-NADH complex was determined to be 2.99 × 10^?6m which was in good agreement with the value obtained from the fluorescence titration experiments.     

Enoyl-ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic mechanism and inhibition by triclosan and hexachlorophene

Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC(50) values for triclosan in the presence and absence of NAD(+) were 0.1 +/- 0.02 and 2.4 +/- 0.02 microM, respectively, confirming previous observations that the E-NAD(+) complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E-NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 microM triclosan to E, E-NADH, E-NAD(+), and E-crotonyl-CoA occur at rates of 0.0124 +/- 0.001, 0.0663 +/- 0.002, 0.412 +/- 0.01, and 0.0069 +/- 0.0001 s(-1), respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD(+)- and time-independent; and the calculated IC(50) value was 2.5 +/- 0.4 microM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E-NAD(+), and E-crotonyl-CoA have similar affinity for hexachlorophene, since the k(obs)s were in the same range of 20-24 s(-1). When the E-NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.     

Identification of essential arginyl residues in cytoplasmic malate dehydrogenase with butanedione.

The inactivation of cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) from porcine heart and the specific modification of arginyl residues have been found to occur when the enzyme is inhibited with the reagent butanedione in sodium borate buffer. The inactivation of the enzyme was found to follow pseudo-first order kinetics. This loss of enzymatic activity was concomitant with the modification of 4 arginyl residues per molecule of enzyme. All 4 residues could be made inaccessible to modification when a malate dehydrogenase-NADH-hydroxymalonate ternary complex was formed. Only 2 of the residues were protected by NADH alone and appear to be essential. Studies of the butanedione inactivation in sodium phosphate buffer and of reactivation of enzymatic activity, upon the removal of excess butanedione and borate, support the role of borate ion stabilization in the inactivation mechanism previously reported by Riordan (Riordan, J.F. (1970) Fed. Proc. 29, Abstr. 462; Riordan, J.F. (1973) Biochemistry 12, 3915-3923). Protection from inactivation was also provided by the competitive inhibitor AMP, while nicotinamide exhibited no effect. Such results suggest that the AMP moiety of the NADH molecule is of major importance in the ability of NADH to protect the enzyme. When fluorescence titrations were used to monitor the ability of cytoplasmic malate dehydrogenase to form a binary complex with NADH and to form a ternary complex with NADH and hydroxymalonate, only the formation of ternary complex seemed to be effected by arginine modification.     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Plant succinic semialdehyde dehydrogenase: dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy

Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we used two approaches to investigate the interaction of ATP with At-SSADH1. Cofactor displacement studies based on the reduced fluorescence intensity of free NADH versus that of enzyme-bound NADH revealed that both AMP and ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH from its binding site. To assess the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to monitor nucleotide binding to immobilized At-SSADH1. For this, we used a Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD(+) and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD(+). GTP competed with ATP. However, only ATP increased the dissociation constant of NAD(+) from SSADH. This explains the reduced affinity of NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic kinetics, and supports our model of feedback regulation of SSADH and the GABA shunt by ATP.     

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ=0.4 ns) and bound NADH (τ=6.0 ns). We used this technique to investigate the effects of Mg(2+) on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K(D) for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg(2+) ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 μM Mg(2+)) to 8.3s(-1) (0 μM Mg(2+)) as determined by addition of excess NAD(+) to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 μM(-1)s(-1) over the entire Mg(2+) ion concentration range and demonstrate that Mg(2+) ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg(2+) concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ=11.2 ns).