MECHANISM OF ALDEHYDE OXIDATION CATALYZED BY HORSE LIVER ALCOHOL-DEHYDROGENASE

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed b y horse liver alcohol dehydrogenase (HLADH) has been investigated usin g the HLADH structure at 2.1 Angstrom resolution with NAD(+) and penta fluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Bioch emistry 33, 5230-5237]. Constructs for molecular dynamics (MD) investi gations with HLADH were obtained by a best-fit superimposition of benz aldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II) ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(lI) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initro -derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21, 40-56], Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MDI) enzyme with NAD(+), benzaldehyde, and zinc-ligated HO- in the active site; ( MD2) enzyme with NAD(+) and hydrated benzaldehyde monoanion bound to z inc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD(+) and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxy gen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempi rical PM3 calculations. Using PM3. model systems were calculated of gr ound states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc -bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resid ed at a distance of 2.8-5.5 Angstrom from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 trans ition state for attack of the Zn(II)-OH oxygen on the benzaldehyde car bonyl carbon, which has an O ... C distance of 1.877 Angstrom. HLADH c atalysis of tile aldehyde hydration would require very little motion a side from that in the ground state. Two simulations of benzaldehyde hy drate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD(+) C4, varying from 2.3 to 3.3 Angstr om, seemingly favorable for the hydride transfer reaction. The MD2 con figuration does not allow proton shuttling. On the other hand, when th e pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxyge n averages 2.09 Angstrom from the hydroxyl oxygen of Ser48 such that i nitiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favor able. PM3 calculations suggest that this proton shuttle represents a s tepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration h as the transferring hydride 1.476 Angstrom from C4 of NAD(+) and 1.433 Angstrom from the aldehyde alpha-carbon.