Dynamic structures of horse liver alcohol dehydrogenase (HLADH): Results of molecular dynamics simulations of HLADH-NAD(+)-PhCH2OH, HLADH-NAD(+)-PhCH2O-, and HLADH-NADH-PhCHO

Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following th ree states have been studied: HLADH.PhCH2OH.NAD(+) (MDI), HLADH.PhCH2O-.NAD (+) (MD2), and HLADH.PhCHO.NADH (MD3). MDI, MD2, and MD3 simulations were c arried out on one of the subunits of the dimeric enzyme covered in a 32-Ang strom -radius sphere of TlP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH2OH.NAD(+) --> HLADH. PhCH2O-.NAD(+) is transferred from the active site to solvent water via a h ydrogen bonding network consisting of serine48 hydroxyl, ribose 2 '- and 3 ' -hydroxyl groups, and Hist51. Hydrogen bonding of the 3 ' OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to wat er. Molecular dynamic simulations have been employed to track water1287 fro m the TIP3 water pool to the active site, thus exhibiting the mode of entra nce of water to the active site. With time the water 1287 accumulates in tw o different positions in order to accept the proton from the ribose 3 ' -OH and from His51. There can be identified two structural substates for proto n passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose CY-OH and hydrogen bonding distances for proton tra nsfer through the hydrogen bonded relay series PhCH(2)OH(...)Ser48-OH(...)R ibose2 ' -OH(...)His51(...)OH(2) (path 1) average 2.0, 2.0, and 2.1 Angstro m and (for His51(...)OH(2)) minimal distances less or equal to 2.5 Angstrom . The structure for path 1 is present 20% of the time span. And in the seco nd substate, there are two possible proton passages: path I as before and p ath 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH(...)Ser48 -OH(...)Ribose2 ' -OH(...)Ribose3 ' -OH(...)His51(...)OH(2) with the averag e bonding distances being 2.0, 2.0, 2.1, and 2.0 Angstrom and (for His51(.. ..)OH(2)) Minimal distances less or equal to 2.5 A (20% probability of the time span), respectively. During the molecular dynamics simulation the NAD( +) ribose conformations have stabilized at the C2 ' -endo-C3 ' -exo or the C2 ' -endo conformations. With the C2 ' -endo conformation the first and se cond substates are able to persist for different time spans, while with the C2 ' -endo-C3 ' -exo conformation the only possible pathway involves the f irst substate, For both first and second substates the fluctuation of the d istances between the ribose-OH protons and N epsilon2 of His51 imidazole ri ng is partially contributed by the "windshield wiper" motion of the His51 i midazole ring. Since the imidazole of His-51 contributes only about 10-fold to activity, as estimated from the decrease in activity upon substitution with a Gln, there must be an alternate route for the proton to pass to solv ent without going through this histidine. A third pathway involves ribose C 3 ' -OH and Ile-269. In MD2, near attack conformers (NACs) for hydride tran sfer from PhCH2O- to NAD(+) represent similar to 60% of E-S conformers. The molecular dynamic study of MD3 at mildly basic pH reveals that reactive gr ound state conformers (NACs) for hydride transfer from NADH to PhCHO amount to 12 mol % of conformers. In MD3, anisotropic bending of the dihydronicot inamide ring of NADH (average value of alpha (c) = 4.0 degrees and alpha (n ), = 0.5 degrees, respectively) is observed.