INTRODUCTION:

Horse liver alcohol dehydrogenase (LADH)1 catalyzes the first, and rate limiting step in alcohol metabolism: the oxidation of alcohol to aldehyde. This reaction requires the transfer of a hydride ion from the alcohol substrate to the cofactor NAD. Despite the fact that LADH is one of the most extensively studied enzymatic systems to date, the mechanism of the hydride transfer step remains an area of active investigation. A quantum mechanical tunneling contribution to this hydride transfer step has been demonstrated by Judith Klinman and her group in the Dept. of Chemistry at UC Berkeley. Klinman and co-workers have observed kinetic isotope effects consistent with tunneling in a number of LADH mutants.

LADH mutants have been designed to test the tunneling hypothesis. Mutant design exploits the hypothesis that thermal fluctuations in protein conformation enhance tunneling by shortening the distance between donor and acceptor atoms. In LADH, the active site is sandwiched between two domains, such that thermally-induced inter-domain motion would compress the distance between the nicotinamide ring and substrate. The back face of the nicotinamide ring makes hydrophobic contact with the highly conserved residue Val 203. Thus, this residue could transmit kinetic energy associated with inter-domain motion to the nicotinamide ring, and ultimately the hydride transfer reaction. Mutation of Val 203 to a less bulky alanine was expected to reduce tunneling by diminishing this potential compression effect. Isotope effects observed in the V203A mutant are consistent with reduced tunneling.

In order to test the structural assumptions behind these mutations, we examined crystal structures of ternary complexes of the LADH active site single mutants F93W and V203A. The single F93W mutant displays isotope effects characteristic of a significant tunneling contribution, with a catalytic efficiency comparable to that of the wild type enzyme. The relative conformation of the cofactor and trifluoroethanol substrate analogue in the F93W mutant structure does in fact approximate that of an active ternary complex in the closed conformation. In contrast, the V203A mutant shows reduced tunneling and an almost 40-fold reduction in catalytic efficiency relative to F93W. The crystal structure of the V203A mutant shows a compromised catalytic site geometry, the NAD nicotinamide ring rotating away from the substrate, toward the gap left by replacement of the adjacent bulky valine at position 203 with the smaller alanine. This increases the distance for hydride transfer in the ground state structure, and accounts for the reduced tunneling seen in this mutant.

RESULTS:

The active site structure from the "high" tunneling (Phe93-Trp mutant of LADH. The Electron density omit map (Fo-Fc, 2.5 sigma) and resulting models for the residue at position-203 (green), NAD+ (C-purple, O-red, P-white, N-blue), and trifluoroethanol (C-yellow, F-orange, O-red) are illustrated. The omit map is generated with trifluoroethanol, NAD+, and residue-203 omitted from the final model. The nicotinamide ring is in van der Waals contact with a methyl group of Val203. The average donor to acceptor carbon distance among the two independent monomers is 3.2 Å, approximating an active ternary complex.

Recently, we have also examined two ternary complexes of the double mutant F93W/V203A. The double mutant shows a 75-fold reduction in catalytic efficiency relative to the native enzyme, and reduced tunneling relative to either single mutant. Comparison of active site structures of complexes between NAD, trifluoethanol and three LADH mutants is shown above. The nicotinamide ring in F93W is in van der Waals contact with Val203. In both V203A and F93W/V203A, the nicotinamide ring rotates (curved arrow) to fill the gap left by the substitution to alanine. Further rotation of the rings is prevented by steric contacts with Trp93 for F93W/V203A and by Thr178 for both mutants (not shown).

(F93W (cyan), F93W/V203A (magenta) and V203A (grey). The trifluoethanol substrate analogue is at left. Overlaps of the two (F93W, F93W/V203A) or four (V203A) crystallographically independent NAD nicotiname rings are at center, viewed edge-on along the glycosidic bond. The side chain of residue 203 is at right. Van der Waals surfaces are drawn for the F93W and F93W/V203A mutants. Nicotinamide overlaps are based on a least squares allignment of the eight independent cofactor binding domains from the three mutants.)

SUMMARY

Structures of the single and double mutants suggest that active-site mutations at the domain interface 1) destabilize closure of the enzyme to the active form, 2) perturb catalytic geometry in the closed conformation and 3) inhibit vibrationally induced enhancement of catalysis. For additional information, see the publications listed below.

Bahnson, B. J., Colby, T. D., Chin, J., Goldstein, B. M., Klinman, J. P. A link Between Protein Structure and Enzyme Catalyzed Hydrogen Tunneling. Proc. Nat. Acad. Sci. , 94: 12797-12802 (1997) . REPRINT *

Colby, T. D.; Bahnson, B. J., , Chin, J., Klinman, J. P.; Goldstein, B. M. Active Site Modifications in a Double Mutant of Liver Alcohol Dehydrogenase: Structural Studies of Two Enzyme-ligand Complexes. Biochemistry , 37:9295-9304 (1998) . REPRINT*

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