A link between protein structure and enzyme catalyzed hydrogen tunneling

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.

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

“…Klinman and coworkers have provided clear evidence of NQM in several enzymes, 15,[134][135][136][137][138] and these findings have frequently been interpreted as evidence that the width of the barrier for tunneling is narrowed by particular vibrations. 139 The identification of large tunneling was used to argue that NQM is important for catalysis (see, e.g., Refs.…”

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

“…In the procedure, it is assumed that the Swain-Schaad relationship holds or has little temperature dependence for intrinsic primary KIEs (Northrop 1991;Francisco et al 2002;Kohen 2003;Cleland 2005). In systems where non-classical isotope effects have been suggested from temperature dependency or theoretical simulation, the experimental relationship between primary H/T and D/T KIEs has been shown to be very close to the semi-classical limit (Northrop 1991;Jonsson et al 1994;Bahnson et al 1997;Chin & Klinman 2000). Several recent gas-phase calculations for a small model reaction also resulted in a Swain-Schaad exponent that did not deviate significantly from its semi-classical value at ambient temperature (300-350 K; Kiefer & Hynes 2003Tautermann et al 2004;.…”

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

“…The reactions catalysed by the alcohol dehydrogenases from yeast, horse liver and Bacillus stearothermophilus have provided some of the most direct evidence for aberrant Swain-Schaad relationships that lie outside these semi-classical limits (Cha et al 1989;Bahnson et al 1997;Kohen et al 1999;table 1). It is noteworthy that the summarized data have been observed for the secondary (non-transferred) hydrogen of alcohol substrates doubly labelled at the methylene position with either LZD, T or H, T; i.e.…”

“…and (iii) enzyme forms that have been mutated at their active sites (table 2; Cha et al 1989;Rucker et al 1992;Bahnson et al 1993Bahnson et al , 1997Kohen et al 1999). The latter involves either an increase in bulk at Leu57 or Phe93 (numbering refers to the horse liver alcohol dehydrogenase (LADH)), to alter the rate of substrate release from the active site pocket and make chemistry more rate determining, or a reduction in bulk at Val203 to alter its interactions with the C-4, reactive position of cofactor.…”