A LINK BETWEEN PROTEIN-STRUCTURE AND ENZYME-CATALYZED HYDROGEN TUNNELING

We present evidence that the size of an active site side chain may mod ulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction , Primary and secondary k(H)/k(T) and k(D)/k(T) kinetic isotope effect s have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 degrees C. As reported in earl ier studies, the relationship between secondary k(H)/k(T) and k(D)/k(T ) isotope effects provides a sensitive probe for deviations from class ical behavior. In the present work, catalytic efficiency and the exten t of hydrogen tunneling have been correlated for the alcohol dehydroge nase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofa ctor NAD(+) from the alcohol substrate, The reduction in size of this residue is correlated with diminished tunneling and a two orders of ma gnitude decrease in catalytic efficiency. Comparison of the x-ray crys tal structures of a ternary complex of a high-tunneling (Phe-93 --> Tr p) and a low-tunneling (Val-203 --> Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydro gen transfer distance for the low-tunneling mutant, The Val-203 --> Al a ternary complex crystal structure also shows a hyperclosed interdoma in geometry relative to the wild-type and the Phe-93 --> Trp mutant te rnary complex structures, This demonstrates a flexibility in interdoma in movement that could potentially narrow the distance between the don or and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.