Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
3alpha-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 (D)V and (D(2)O)V for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, (2)H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys(159) changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on V(max) and V/K(androsterone) and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on V(max) 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 Brønsted relationship between the log(V/K(d)(-base)Et) of the external amine (corrected for molecular size effects) and pK(a) is linear for the K159A mutant-catalyzed reaction at pH 10.4 (beta = 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 Tyr(155) to Lys(159) via 2'-OH of ribose.
Chang
Yi-Hsun
YH
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan.
Chuang
Lea-Yea
LY
Hwang
Chi-Ching
CC
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D017738:Alkanesulfonic Acids / Q000737:chemistry; D019943:Amino Acid Substitution; D000738:Androsterone / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics; D003514:Cyclohexylamines / Q000737:chemistry; D005043:Etiocholanolone / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009243:NAD / Q000737:chemistry; D011522:Protons
D017738:Alkanesulfonic Acids; D001426:Bacterial Proteins; D003514:Cyclohexylamines; D011522:Protons; D009243:NAD; C097300:3-(cyclohexylamino)-1-propanesulfonic acid; C006576:androstane-3,17-dione; D005043:Etiocholanolone; D000738:Androsterone; C425306:3alpha-hydroxysteroid dehydrogenase-carbonyl reductase; D000429:Alcohol Oxidoreductases; D006913:Hydroxysteroid Dehydrogenases