Hydrophobic interactions of a substrate with L193 on the flexible substrate-binding loop contribute to...

Flexible loops within enzyme active sites are central to substrate binding, transition-state stabilization, and catalytic turnover. Hydrophobic interactions modulate ligand recognition and influence protein function. Here, we interrogated loop-mediated hydrophobic contributions to catalysis in 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni. Residue L193 on the flexible substrate-binding loop was substituted with amino acids spanning a hydrophobicity gradient (W, I, F, M, V, P, A, T, G). Differential scanning fluorimetry showed comparable melting temperatures among variants, indicating minimal effects on global thermal stability. Steady-state kinetics revealed decreased catalytic efficiency with reduced side-chain hydrophobicity and corresponding increases in activation free energy (ΔG^‡). With the exception of L193W, L193I, L193F, L193P, and L193T, log(k_cat/K_M) correlated linearly and positively with residue hydrophobicity, supporting a key role of hydrophobic interactions involving L193 in stabilizing the transition state of androsterone in the 3α-HSD/CR-catalyzed reaction. Truncated substrates (2-decalol, cyclohexanol) cause a significant decrease in catalytic efficiency, exhibiting similar trends with shallower slopes, consistent with predominant L193 close to the steroid A-ring. Molecular dynamics simulations indicated that NAD⁺ and substrate binding promote loop closure and strengthen hydrophobic interactions involving L193 in the active site. These data establish that the hydrophobic interaction of residue 193 with the transition state of substrate, linked to remote binding interactions of the loop with the unreactive portion of the substrate, significantly contributes to 3α-HSD/CR catalysis.