Soybean lipoxygenase catalyzes a proton-coupled electron transfer (PCET) reaction and serves as a prototype for hydrogen tunneling in enzymes due to the unusually high kinetic isotope effect and significant modulation of the rate constant and kinetic isotope effect by mutation. Herein these experimental observations are interpreted in the context of changes to the substrate binding pocket in microsecond molecular dynamics simulations of wild-type and mutant soybean lipoxygenase. The binding pocket exhibits an hourglass shape with residues L546 and L754 bracketing the bottleneck, positioning the linoleic acid substrate for PCET. Mutation of I553 to less bulky residues slightly increases the width of the bottleneck and the volume of the binding pocket. Mutating L546 or L754 to a smaller residue also enlarges this width and volume, and mutating both has an even more dramatic effect. This analysis illustrates how mutation of the substrate binding pocket can be used as a strategy to tune the kinetics.