Mechanism of Proton Transfer in the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

Yi-Hsun Chang,Lea-Yea Chuang,Chi-Ching Hwang

doi:10.1074/jbc.m706336200

Yi-Hsun Chang, Lea-Yea Chuang + Show 1 more

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https://doi.org/10.1074/jbc.m706336200

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Abstract

Abstract 3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of DV and D2OV for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, 2H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys159 changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on Vmax and V/Kandrosterone and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on Vmax is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Bronsted relationship between the log(V/Kd-baseEt) of the external amine (corrected for molecular size effects) and pKa is linear for the K159A mutant-catalyzed reaction at pH 10.4 (β = 0.85 ± 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr155 to Lys159 via 2′-OH of ribose.

Highlights

␤-sheet flanked by ␣-helices [2, 3]
Chemical Rescue in the K159A Mutant Enzyme by Proton Acceptors—In a previous study, the pH profile of the K159A mutant enzyme showed similar pH dependence compared with wild-type enzyme with a shift of the pKa in V/KandrosteroneEt from 7.2 to 9.1, suggesting that Lys159 lowers the pKa of the Tyr155, which acted as a general base
The phenol group of Tyr155 was deprotonated for an optimal reaction in 100 mM CAPS at pH 10.4, the activity of K159A mutant enzyme was 50-fold less than that of the wild-type in V/KandrosteroneEt and 10-fold less in V/Et (Table 1)

Summary

Introduction

␤-sheet flanked by ␣-helices [2, 3]. In the binary complex, the NADϩ cofactor is bound at the carboxyl-terminal ends of the ␤-strands in the 3␣-HSD/CR from C. testosteroni. 3␣-HSD/CR reversibly catalyzes the oxidation of the steroid alcohol using NADϩ as the oxidant. The overall oxidoreductive reaction catalyzed by 3␣-HSD/CR is composed of the deprotonation of tyrosine, proton abstraction by the tyrosinate anion, and hydride transfer from the hydroxysteroid to NADϩ, followed by the release of a proton from the hydroxy group of tyrosine to the solution (Fig. 1). The chemical mechanism of 3␣-HSD/CR is involved in the deprotonation of tyrosine to form tyrosinate anions in the ternary complex (a), and the abstraction of the proton from the 3-hydroxyl group of the androsterone, followed by hydride transfer to form androstanedione and NADH (b) with a concomitant release of proton and products to the bulk solvent (c). In addition to lowering the pKa of the general base Tyr155, the results further demonstrated the role of Lys159 in shuttling the protons to bulk solvent in the 3␣-HSD/CR-catalyzed reaction

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