Although there are considerable data demonstrating that quantum mechanical hydrogen tunneling (HT) occurs in both enzymatic and nonenzymatic systems, little data exist that address the question of whether enzymes enhance the amount of HT relative to the corresponding nonenzymatic reactions. To investigate whether 3-oxo-Delta(5)-steroid isomerase (ketosteroid isomerase, KSI) enhances HT relative to the nonenzymatic (acetate-catalyzed) isomerization of Delta(5)-androstene-3,17-dione (1) to Delta 4-androstene-3,17-dione (3), a-secondary deuterium kinetic isotope effects (KIE) at C-6 of the steroid were determined for both the KSI- and acetate-catalyzed isomerizations. The normal intrinsic secondary KIE for both wild type (WT) KSI (1.073 +/- 0.023) and acetate (1.031 +/- 0.010) suggest the possibility of coupled motion (CM)/HT in both the enzymatic and nonenzymatic systems. To assess the contribution of CM/HT in these reactions, the secondary KIE were also measured under conditions in which deuterium instead of hydrogen is transferred. The decrease in secondary KIE for WT (1.035 +/- 0.011) indicates the presence of CM/HT in the enzymatic reaction, whereas the acetate reaction shows no change in secondary KIE for deuterium transfer (1.030 +/- 0.009) and therefore no evidence for CM/HT. On the basis of these experiments, we propose that KSI enhances the CM/HT contribution to the rate acceleration over the solution reaction. Active site mutants of KSI (Y14F and D99A) yield secondary KIES similar to that of WT, indicating that mutations at the hydrogen-bonding residues do not significantly decrease the contribution of CM/HT to the KSI reaction.