delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereoselective transfer of the 4 beta-proton to the 6 beta-position. The rate-limiting step has been shown to be the concerted enolization of the enzyme-bound substrate comprising protonation of the 3-carbonyl oxygen by Tyr-14 and abstraction of the 4 beta-proton by Asp-38 [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. Primary, secondary, solvent, and combined kinetic deuterium isotope effects have been used to investigate the mechanism of the Y14F mutant, which lacks the proton donor and is 10(4.7)-fold less active catalytically than the wild-type enzyme. With [4 beta-D]androst-5-ene-3,17-dione as a substrate in H2O, a lag in product formation is observed which approaches, by a first-order process, the rate observed with protonated substrate. With the protonated substrate in D2O, a burst in product formation is detected by derivative analysis of the kinetic data which approaches the rate observed with the 4 beta-deuterated substrate in D2O. The absence of such lags or bursts with the protonated substrate in H2O or with the 4 beta-deuterated substrate in D2O, as well as the detection of buffer catalysis by phosphate at pH 6.8, indicates that one or more intermediates dissociate from the enzyme and partition to substrate 31.6 times faster than to product.(ABSTRACT TRUNCATED AT 250 WORDS)