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 k_H/k_T and k_D/k_T kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 deg C. As reported in earlier studies, the relationship between secondary k_H/k_T and k_D/k_T 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.
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