Determinants of the rate-limiting events in 3α-hydroxysteroid dehydrogenase (3α-HSD) catalysis

Hydroxysteroid dehydrogenases (HSDs) control local steroid hormone levels in steroid target tissues by converting potent metabolites into their inactive form. Rat liver 3α-HSD (AKR1C9) provides a solid model to address the mechanistic issues of steroid transforming enzymes. The ternary complex structure of AKR1C9· NADP+· testosterone provided an excellent opportunity to understand the role of steroid-binding pocket residues to steroid affinity and turnover. Using alanine scanning mutagenesis, it was shown that conserved residues play a significant role in hydroxysteroid oxidation. Ligand binding and steady state kinetic analyses showed negligible effects in Kd and Km, but dramatic effects in kcat. Transient kinetic analysis showed that the rate-determining step in androsterone oxidation was altered from product release in AKR1C9 to chemistry in mutants with significantly low kcat. These findings suggest that binding pocket residues dictate optimal substrate positioning and validate the importance of proximity in enzyme catalysis. 3α-HSDs eliminate the androgen signal by converting 5α-DHT, a potent androgen to 3α-diol, a weak androgen. Using AKR1C9 and these physiologically relevant substrates, transient kinetics and kinetic isotope effect (KIE) studies showed that the rate-limiting event is chemistry in the NADPH-dependent reduction of 5α-DHT and product release in the NADP +-dependent oxidation of 3α-diol. When Arg276, which interacts with the 2′-AMP of NADP(H), and Trp227, which makes van der Waals contact with steroid, were mutated, the chemical step became rate-limiting in steroid oxidation. The chemical step was also rate-determining when NAD + was utilized. Because the major cellular cofactors are NADPH and NAD+, the chemical step was identified as rate-limiting in both directions. AKR1C2 is the most pertinent human 3α-HSD as it eliminates 5α-DHT in peripheral tissues. The rate-determining event in AKR1C2 was compared to AKR1C9 using KIEs to determine whether they catalyze similar or different rate-determining steps. Using the substrate pairs: 5α-DHT/3α-diol and androstanedione/androsterone, comparative analyses showed that while AKR1C2 displayed depressed kcat values, the rate-limiting event is not the chemical step as expected. Instead a combination of slow steps contributes to the low rates observed. Taken together, HSDs, which are closely related with high sequence identities, have different microscopic events that govern the overall rate-determining step