Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1–1.3 Å resolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13–16 Å2) over the range 25–100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7–10 Å2 for the overall complexes and of 5–10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Å and is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

PDB references: alcohol dehydrogenase, complex with NAD and TFE, 25 K, 7ua6; 45 K, 7uc9; 65 K, 7uca; 85 K, 7ucu; 100 K, 4dxh; 125 K, 7ude; 150 K, 7udd; complex with NAD and PFB, 25 K, 7udr; 50 K, 7uec; 75 K, 7uee; 85 K, 7uef; 100 K, 7uei; 150 K, 7uej; G173A alcohol dehydrogenase, complex with NAD and PFB, 50 K, 7uhv; 85 K, 5kj1; 120 K, 7uhw; 150 K, 7uhx