Fast Motions in 5 Alpha Reductase and Its Impact on Enzyme Kinetics.

Behind the catalytic efficiency of enzymes lies a finely tuned dynamic interplay among residues that cooperatively orchestrate the reaction. Steroid 5α-reductase type 2 (SRD5A2) catalyzes NADPH-dependent reduction of testosterone to dihydrotestosterone through sequential hydride and proton transfer. Our study addresses fundamental gaps in understanding the catalytic mechanism-activation barriers, rate-promoting dynamics, and electrostatic contributions-none of which have been characterized to date. Using QM/MM simulations with transition path sampling, we have shown how molecular motions can impact the kinetics of a catalytic reaction. We have found that two residues, Tyr33 and L224, have a compression effect on the donor, which not only brings a significant change in the free energy barriers but can also perturb the local electric field, which supports the preorganization theory. Along with that, we have found, through extensive committor analysis, 6 additional residues, Trp53, Arg94, Cys119, Glu197, Phe223, and Arg227, that constitute an extended reaction coordinate network, stabilizing transition states through coupled electrostatic and structural interactions. Analysis of the disease-associated L224P mutant reveals that loss of the L224 compression eliminates field enhancement and increases barriers by 3.3-3.4 kcal/mol, establishing that efficient catalysis requires temporal orchestration of dynamics and electrostatics across the extended protein architecture.