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.

Primary and secondary kD/kT and kH/kT kinetic isotope effects have been studied as a probe of hydrogen tunneling in the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase (LADH). In the case of the wild-type enzyme, isotope effects at 25 degrees C do not clearly support hydrogen tunneling; this result is consistent with a reaction rate that is partially limited by the release of product benzaldehyde. The three-dimensional structure for LADH was used to design site-directed mutations in an effort to enhance the rate of the product release step and to "unmask" tunneling. Substitutions that increased the size of the alcohol binding pocket resulted in minor changes in isotope effects. By contrast, reduction in the size of the alcohol binding pocket through substitution at residues 57 and 93, which are in van der Waals contact with bound alcohol substrate, produced a clear demonstration of protium tunneling from the breakdown of the semiclassical relationship between kD/kT and kH/kT isotope effects. The temperature dependence of kD/kT isotope effects has also been pursued, leading to the conclusion that tunneling does, in fact, occur in the reaction catalyzed by wild-type LADH. Despite the unmasking of protium tunneling in site-directed mutants, substitutions that decrease the size of the alcohol pocket appear to result in less extensive tunneling in the hydride transfer. It is noteworthy that the mutant enzyme (Leu57-->Phe), which shows the greatest evidence of tunneling, has the same catalytic efficiency (Vmax/Km) as the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10 13 s −1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.A complete understanding of the origins of the remarkable rate acceleration and specificity achieved by enzymes remains elusive. Increasingly, studies on the fundamental basis for enzymatic catalysis have focused on the requirement for a range of protein motions in order to achieve efficient enzyme catalysis. Among these, evidence for a class of motion termed conformational sampling (or preorganization) has begun to accumulate recently (cf. refs. 1-4). The impact of such motions can be seen either in the formation of an enzyme-substrate complex or in its subsequent conversion to product.For example, a recent study of adenylate kinase implicates a temperature-dependent preequilibrium among conformers that are either "competent" or "incompetent" toward ligand binding (5). In the context of catalysis, studies of single-enzyme molecules indicate multiple, slowly interconverting conformers that lead to product with different rate constants (6-8). Extensive work on dihydrofolate reductase has shown the role of slow loop closures that occur along the reaction coordinate with the same (millsecond) time constants as catalysis (9, 10).Although computational studies also suggest a role for rapid conformational sampling in enzyme reactions (11, 12), demonstrating the direct link of these motions to catalysis is quite challenging. In this study, we use the thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) as a system to examine the presence of rapidly interconverting protein substates during catalysis. The ht-ADH catalyzes the transfer of a hydride equivalent from an alcohol donor to an NAD þ cofactor, and previous studies have shown that ht-ADH carries out its reaction via quantum t...

The crystal structure of porcine heart mitochondrial NADP ؉ -dependent isocitrate dehydrogenase (IDH) complexed with Mn 2؉ and isocitrate was solved to a resolution of 1.85 Å. The enzyme was expressed in Escherichia coli, purified as a fusion protein with maltose binding protein, and cleaved with thrombin to yield homogeneous enzyme. The structure was determined by multiwavelength anomalous diffraction phasing using selenium substitution in the form of selenomethionine as the anomalous scatterer. The porcine NADP ؉ -IDH enzyme is structurally compared with the previously solved structures of IDH from E. coli and Bacillus subtilis that share 16 and 17% identity, respectively, with the mammalian enzyme. The porcine enzyme has a protein fold similar to the bacterial IDH structures with each monomer folding into two domains. However, considerable differences exist between the bacterial and mammalian forms of IDH in regions connecting core secondary structure. Based on the alignment of sequence and structure among the porcine, E. coli, and B. subtilis IDH, a putative phosphorylation site has been identified for the mammalian enzyme. The active site, including the bound Mn 2؉ -isocitrate complex, is highly ordered and, therefore, mechanistically informative. The consensus IDH mechanism predicts that the Mn 2؉ -bound hydroxyl of isocitrate is deprotonated prior to its NADP ؉ -dependent oxidation. The present crystal structure has an active site water that is well positioned to accept the proton and ultimately transfer the proton to solvent through an additional bound water.