Revised Manuscript Received July 11, 1995® abstract: A series of kinetic isotope effect experiments were performed with the goal of understanding the nature of rate-limiting steps in the soybean lipoxygenase-1 (SBL-1) reaction. SBL-1 was reacted with linoleic acid (LA) and deuterated linoleic acid (D-LA) under a variety of experimental conditions involving changes in temperature, pH, viscosity, and replacement of H2O with D2O. The extrapolated intrinsic primary H/D isotope effect can be estimated to be possibly as large as 80. This value is probably the largest isotope effect published for an enzymatic reaction, and much larger than that predicted from semiclassical models. Due to this large primary isotope effect, the C—D bond cleavage fully limits the rate of reaction under all conditions tested. In the case of protonated linoleic acid, a number of steps are partially rate-limiting at room temperature; three distinct mechanistic steps which include substrate binding, an H2O/D2O sensitive step, and C—H bond cleavage have been characterized. Use of glucose as a solvent viscosogen demonstrates that substrate binding is approximately 48% rate-limiting for LA at 20 C. SBL-1 is one of the few enzymes that fit the definition of a “perfect enzyme”, in the sense that further optimization of any single step at room temperature will not significantly increase the overall rate. At lower temperatures, the step sensitive to solvent deuteration begins to dominate the reaction, whereas at higher temperatures, the hydrogen abstraction step is rate-limiting. The pH dependence of kcJKm for SBL-1 can be explained as arising from two pKfs, one controlling substrate binding and the other substrate release. Below pH 7.8, the rate of substrate release increases, thus decreasing the commitment to catalysis and unmasking the large intrinsic isotope effect on the subsequent hydrogen abstraction. An abnormally high p, in the range of 7—8, has been determined for LA in the concentration range employedin these studies. We propose that the negatively charged form of LA, predominating above pH 7.8, is the preferred substrate with larger commitments to catalysis.