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. 1997 Nov 25;94(24):12797-802.
doi: 10.1073/pnas.94.24.12797.

A link between protein structure and enzyme catalyzed hydrogen tunneling

B J Bahnson  1 , T D Colby, J K Chin, B M Goldstein, J P Klinman
Affiliations

A link between protein structure and enzyme catalyzed hydrogen tunneling

B J Bahnson et al. Proc Natl Acad Sci U S A. .

Abstract

We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 degrees C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 --> Trp) and a low-tunneling (Val-203 --> Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 --> Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 --> Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.

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Figures

Figure 1
Figure 1
Correlation of the log(kcat/Km) and the ratio of ln(kH/kT)/ln(kD/kT) for site-directed mutants of alcohol dehydrogenase. Point A, Val-203 → Gly; point B, Val-203 → Ala:Phe-93 → Trp; point C, Val-203 → Ala; point D, Val-203 → Leu; point E, Phe-93 → Trp; point F, Leu-57 → Phe. Tunneling is indicated when the ratio of ln(kH/kT) to ln(kD/kT) is >3.3 (8).
Figure 2
Figure 2
Comparison of active site structures from a high tunneling (Phe-93 → Trp, top) and a low tunneling (Val-203 → Ala, bottom) mutant of LADH. Electron density omit maps (Fo − Fc) 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 for each structure. Omit maps were generated with trifluoroethanol, NAD+, and residue-203 omitted from the final model and are contoured at σ levels of 2.5 (top) and 2.0 (bottom). (Upper): The nicotinamide ring in Phe-93 → Trp is in van der Waals contact with a methyl group of Val-203. The average donor to acceptor carbon distance among the two independent monomers is 3.2 Å. (Lower): In Val-203 → Ala, van der Waals contact between residue 203 and the nicotinamide ring is removed, causing a shift in ring position (red arrow, see Fig. 3). The average donor to acceptor carbon distance among the four crystallographically independent monomers is 4.0 Å. [Figs. 2 and 4 were rendered using molscript (24), rayscript (E. Fontana, D. Peisach, and E. Peisach, Brandeis University) and rayshade (version 4.0, C. Kolb and R. Bogart, Princeton University).]
Figure 3
Figure 3
Comparison of the four crystallographically independent nicotinamide rings in Val-203 → Ala (green) with the two independent rings in Phe-93 → Trp (blue). Surfaces are drawn at ≈90% of the van der Waals radii. Overlap of the structures is based on a least-squares alignment of the six independent cofactor binding domains. The nicotinamide rings are viewed edge-on along the glycosidic bond from the ribose, approximately normal to the view shown in Fig. 2. The nicotinamide ring in Phe-93 → Trp is in van der Waals contact with the “upper” methyl group of Val-203. In Val-203 → Ala, the nicotinamide ring rotates (curved arrow) to fill the gap left by replacement to alanine (straight arrow). Further rotation of the ring is prevented by steric contacts with Thr-178 (data not shown). Trifluoroethanol is displayed from the Phe-93 → Trp structure.
Figure 4
Figure 4
Difference in interdomain conformation between the hyperclosed Val-203 → Ala LADH structure and the closed Phe-93 → Trp structure. Cofactor domains at left were aligned by a least-squares fit of the two crystallographically independent Phe-93 → Trp monomers (red) and the four independent Val-203 → Ala monomers (white). The hyperclosed conformation of the Val-203 → Ala mutant results from an ≈0.5-Å average rigid body translation of its catalytic domain toward its coenzyme domain, when compared with the closed conformation of the Phe-93 → Trp structure. Vectors describing the rigid body translation for each Val-203 → Ala monomer correlate well, all lying within 25° of the average translation vector (arrow). Rotational components of this motion range between 0.5 and 2.5°, but there is little directional correlation between rotation axes for each of the four independent monomers. Analysis of interdomain motion used CCP4 least-squares fitting routine Lsqkab (32, 33). Val-203 (green), NAD+ (purple), Trp-93 (blue), and trifluoroethanol (C, yellow; F, orange; O, red) are displayed from the Phe-93 → Trp structure.
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