Global conformational change when ADH binds coenzyme. The dimer in red is the apoenzyme conformation (1JU9.pdb [65]), and the dimer in green is the holoenzyme with the zincs and coenzyme shown in space-filling representation (1HLD.pdb [20]). The coenzyme binding domains (Coe) are superimposed. The major change is apparent from the shifts in the catalytic domains (Cat). The loop containing residues 292 to 299 (290's) is indicated. The figures were prepared with Molray [109].

Binding of 2,3,4,5,6-pentafluorobenzyl alcohol in the active site of horse liver alcohol dehydrogenase. Water is excluded from the site of hydride transfer. This complex has an orientation that resembles the presumed Michaelis complex. The figure is based on 1HLD.pdb [20]. (Stereo image is for cross-eyed viewing.)

Structure of the horse liver alcohol dehydrogenase complexed with NADH and N-isopropylformamide. Note that the nicotinamide ring is puckered. The dotted black line connects C4 of the nicotinamide ring to C1 of the formamide to illustrate hydride transfer (3.6 Å). The figure is based on 1P1R.pdb [23].

Transient and simulated progress curves for reaction of ADH with NAD⁺ and caprate. Points are experimental and lines are simulated progress curves for absorbance at 559 nm due to proton release and uptake with phenol red as indicator (A, B), protein fluorescence (C, D), and absorbance at 280 nm (E, F). Enzyme was allowed to react with two different concentrations of caprate and three or four different concentrations of NAD⁺ in the three different exxperiments. The lines are calculated using estimated microscopic rate constants for the mechanism in Scheme 3. The reactions were carried out in 33 mM Na₂SO₄ and 0.25 mM EDTA, pH 8.0 at 25° C. (Reprinted with permission from Kovaleva, E. G., and Plapp, B. V. (2005) Biochemistry 44, 12797-12808 [75]. Copyright 2005 American Chemical Society.)

Since the binding of NAD⁺ results in the conformational change, and His-51 is involved in the process, it was of interest to investigate which groups control the binding of NAD⁺. The equilibrium dissociation constant (equivalent to K_ia) shows highest affinity at high pH and lower affinity at low pH [68]. The rate constants for binding of NAD⁺ show a bell-shaped pH dependence with pK values of about 7 and 9 [35, 44]. One interpretation of these results is that a group on the free enzyme, perhaps the zinc-water, with a pK of 9 is shifted to a pK of 7 in the enzyme-NAD⁺ complex [69-71]. However, X-ray crystallography shows that His-51 and Lys-228 participate directly in binding of NAD⁺, and their contributions to the pH dependence were therefore investigated by using the H51Q and K228R enzymes [72]. The H51Q substitution causes a large effect, decreasing the rate constant for binding of NAD⁺ at pH 8 from 1.2 μM⁻¹s⁻¹ to 0.09 μM⁻¹s⁻¹ and producing a pH dependence with maximal rates above a pK of 8.0. The doubly mutated enzyme, H51Q/K228R, is similar, but the rate constant decreases to 0.011 μM⁻¹s⁻¹ and the pK value increases to 9.0. In the doubly mutated enzyme, it seems likely that the ionization of the catalytic zinc-water is controlling NAD⁺ binding. His-51 appears to facilitate the deprotonation as shown in Scheme 2. The ligand to the zinc can be water or alcohol, and in the absence of NAD(H) in the apoenzyme, a water molecule substitutes for the 2′-hydroxyl group (1YE3.pdb). This mechanism is in principle the same as the one proposed by Brändén et al., except for the additional hydroxyl group in the relay [9]. Interestingly, the rate constants and pH dependence for binding of NADH are only modestly affected by these substitutions. The steady-state kinetic constants for the H51Q enzyme are very similar to those of wild-type enzyme [72], and the three dimensional structures of ternary complexes with NAD⁺ and difluorobenzyl alcohols are essentially superimposable with corresponding structures of wild-type enzyme [67]. These studies show that the proton relay system (Scheme 2) is important for controlling the overall binding of NAD⁺, but leave open the question of how the conformational change is controlled.

Although we propose that the proton is released from the zinc-water, His-51 facilitates the process as the H51Q enzyme only binds NAD⁺ about 1/12 as fast as wild-type enzyme does, and the substitution dramatically shifts the pH dependencies. A simple structural explanation is that His-51 facilitates the relay of the proton from the zinc-water to solvent, and the formation of the zinc-hydroxide attracts the positively-charged nicotinamide ring to a position near the zinc in a reaction coupled to the conformational change (Scheme 4). Whether or not the conformational change or the deprotonation occurs first is an open question, but it does not appear likely that the H51Q substitution would hinder the conformational change, whereas it would affect proton transfer. Thus it appears that deprotonation occurs first and controls the global conformational change. The structural change proposed in Scheme 4 is consistent with the observation that for those complexes of modified enzymes with NAD that crystallize in the open conformation, the position of the nicotinamide ring is not apparent in the electron density maps, but in complexes with the closed conformation the nicotinamide ring is next to the zinc (e.g.,refs. [65, 66].

Conformational change of the flexible loop. Residues 291 to 300 from the apoenzyme (1YE3.pdb) are shown in red, and those from holoenzyme (1HLD.pdb) are shown in standard colors. Note that the rearrangement flips the position of Val-294, which forms a floor under the nicotinamide ribose and protrudes into the substrate binding site (PFB, 2,3,4,5,6-pentafluorobenzyl alcohol).

Hydrogen bonded system in the active site. The hydroxyl group of the alcohol is connected (red dashed lines) via Ser-48 and the 2′-hydroxyl group of the coenzyme to His-51, which contacts a solvent water on the surface of the protein (based on 1HLD.pdb). This system could function as a relay system to allow the alcohol to deprotonate and transfer the proton to solvent. See Scheme 2. The black dotted line connects C4 of the nicotinamide ring to C7 of the benzyl alcohol to indicate the hydride transfer (3.4 Å).

Selected amino acid residues in the NAD binding site. Isoleucine residues 224 and 269 make a sandwich of the adenine ring. Lys-228 interacts with the 3′-hydroxyl group of the adenosine ribose and via a water molecule with a phosphate oxygen. Val-292 is near the nicotinamide ring. The figure is based on 1HLD.pdb.

The minimal kinetic mechanism for horse liver alcohol dehydrogenase is an Ordered Bi Bi reaction, with the addition of an isomerization step (step 2) for the enzyme-NAD⁺ complex, which probably reflects the conformational change shown by X-ray crystallography (Scheme 1). (We use “isomerization” to refer to a kinetic event, whereas “conformation” refers to a structural change.)

Since a macroscopic pK value of about 9.2 in apoenzyme is shifted to a pK value of about 7.6 in the enzyme-NAD⁺ complex, accompanied by release of about one proton per subunit [73, 74], proton release could control the conformational change or vice versa. The sequence of events was investigated by transient kinetic studies of proton release and protein fluorescence or UV absorption during the binding of NAD⁺ and formation of ternary complexes [75]. The critical experiments illustrated in Fig. 7 show a biphasic proton release and uptake when caprate and NAD⁺ bind, with varied concentrations of ligands. The families of transient progress curves were fitted by kinetic simulation to provide estimates of the rate constants for each step in the mechanism. Several different kinetic mechanisms were examined, and the simplest mechanism that fits the data is shown in Scheme 3, where a rate-limiting deprotonation of the HE-NAD⁺ complex (k₂, about 230 s⁻¹) is coupled to the conformational change and occurs before the caprate binds. The initial deprotonation is coincident with changes in protein fluorescence and absorption at 280 nm, which seem to report on the protein conformation [60, 76, 77]. In this case, the kinetic “isomerization” is concluded to be equivalent to the structural “conformational change”. The binding of NAD⁺ appears to be in rapid equilibrium (K₁ = 0.67 mM), and pK₂ for the HE-NAD⁺ complex is 7.3. The initial proton release phase is followed by proton uptake (equivalent to hydroxide dissociation) associated with caprate binding, demonstrating that the conformational change occurs before caprate binds. A similar mechanism was obtained for binding of 2,2,2-trifluorethanol and pyrazole, except that they only bind preferentially (about 10 times faster) to the deprotonated E-NAD⁺ complex as compared to HE-NAD⁺, and only a proton release phase is associated with their binding. The simplest interpretation is that the E-NAD⁺ complex has a zinc-hydroxide, and trifluoroethanol and pyrazole deprotonate upon binding to produce a water and the zinc alkoxide or pyrazolate.

An alternative mechanism involves a double displacement in which a glutamate residue that is located on the opposite side of the zinc from which the substrate approaches, moves into ligation with the zinc and displaces the hydroxide, and then is subsequently displaced by the incoming ligand. Such a mechanism was suggested on the basis of theoretical calculations [80] and is illustrated in Scheme 5. (This scheme is based on the residue numbering for yeast alcohol dehydrogenase.) Note that the water and the alcohol are shown in a protonated state, but the water would be deprotonated through the proton relay system before displacement by the glutamate carboxyl group, and after an alcohol binds it would deprotonate to form the alkoxide before transfer of the hydride ion to NAD⁺. This mechanism is supported by X-ray crystallography, where for instance, the homologous dimeric human ADH3 (glutathione-dependent formaldehyde dehydrogenase) shows two different ligation states, which depend upon the ligands [81]. In subunits of the enzyme where the exogenous ligand is water or 12-hydroxydodecanoic acid (a substrate), or NADH and hydroxymethylglutathione (a physiological substrate), the ligand is bound to the tetracoordinated zinc and the glutamate is 4.8 Å away from the zinc. In contrast, in subunits with NAD(H) or NAD⁺ and dodecanoic acid, the glutamate is ligated to the tetracoordinated zinc. It appears that when the ligand is neutral, the glutamate will coordinate to the zinc. In the homologous, but tetrameric, ADH from Escherichia coli the situation is more complicated, as the apoenzyme has three of the four subunits with the alternative coordination with glutamate, whereas the holoenzyme with NAD(H) has 3 of the 4 subunits with the classical coordination with water as a ligand. The fourth subunit has no bound coenzyme and the alternative coordination [3, 82]. In these complexes, the zinc has moved about 2 Å during the conformational change. Tetrameric alcohol dehydrogenases from Clostridium beijerinckii, Sulfolobous solfataricus, and Saccharomyces cerevisiae also have some subunits in which the glutamate is ligated to the zinc [83-85][2hcy.pdb, Plapp, unpublished]. Some experimental support for a role for the glutamate in catalysis comes from a study of yeast alcohol dehydrogenase, in which the substitution of the glutamate with glutamine decreases catalytic efficiency by 100-fold [86]. However, in the Thermoanaerobacter brockii enzyme, substituting the glutamate with alanine or aspartic acid residues only decreases activity by 4 to 6-fold [87]. Although the mechanism of ligand exchange is unknown at this time, it is clear that there is considerable flexibility in the zinc coordination that would make the mechanism shown in Scheme 5 plausible.