Retention of NADPH-Linked Quinone Reductase Activity in an Aldo-Keto Reductase Following Mutation of the Catalytic Tyrosine

Abstract

Aldo-keto reductases (AKR) are monomeric oxidoreductases that retain a conserved catalytic tetrad (Tyr, Lys, Asp, and His) at their active sites in which the Tyr acts as a general acid−base catalyst. In rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD, AKR1C9), a well-characterized AKR, the catalytic tyrosine is Tyr 55. This enzyme displays a high catalytic efficiency for a common AKR substrate 9,10-phenanthrenequinone (9,10-PQ). Surprisingly, Y55F and Y55S mutants of 3α-HSD reduced 9,10-PQ with high k_cat values. This is the first report whereby the invariant catalytic tyrosine of an AKR has been mutated with retention of k_cat values similar to wild-type enzyme. The Y55F and Y55S mutants displayed narrow substrate specificity and reduced select aromatic quinones and α-dicarbonyls. k_cat versus pH profiles for steroid oxidoreduction catalyzed by wild-type 3α-HSD exhibited a single ionizable group with a pK = 7.0−7.5, which has been assigned to Tyr 55. This group was not evident in the k_cat versus pH profiles for 9,10-PQ reduction catalyzed by either wild-type or the Tyr 55 mutant enzymes, indicating that the protonation state of Tyr 55 is unimportant for 9,10-PQ turnover. Instead, wild-type and the active-site mutants Y55F, Y55S, H117A, D50N, K84R, and K84M showed the presence of a new titratable group with a pK_b = 8.3−9.9. Thus, the group being titrated is not part of the tetrad. All the mutants decreased k_cat/K_m considerably more than they decreased k_cat. Thus, the K84R mutant demonstrated a 30-fold decrease in the pH-independent value of k_cat but 2200-fold decrease in the pH-independent value of k_cat/K_m. This suggests that all the tetrad residues influence quinone binding and that Lys 84 plays a dominant role in maintaining proper substrate orientation. Using wild-type enzyme, the energy of activation (E_a) for 9,10-PQ reduction was ∼11 kcal/mol less than steroid oxidoreduction. The E_a for 9,10-PQ reduction was unchanged in the Tyr 55 mutants, suggesting that the reaction proceeds through the same low-energy barrier in the wild-type enzyme and these mutants. The retention of quinone reductase activity in this AKR in the absence of Tyr 55 with k_cat versus pH rate profiles and activation energies identical to wild-type enzyme suggests that quinone reduction occurs via a mechanism that differs from 3-ketosteroid reduction. In this mechanism, the electron donor (NADPH) and acceptor (o-quinone) are bound in close proximity, which permits hydride transfer without formal protonation of the acceptor carbonyl by Tyr 55. This represents a rare example where one enzyme can catalyze the same chemical reaction (carbonyl reduction) by either acid catalysis or by a propinquity effect and where these two mechanisms can be discriminated by site-directed mutagenesis.