PMID- 15379543
OWN - NLM
STAT- MEDLINE
DCOM- 20041109
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 43
IP  - 38
DP  - 2004 Sep 28
TI  - Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by 
      rat 3alpha-HSD via transient kinetics shows that the chemical step is 
      rate-determining: effect of mutating cofactor and substrate-binding pocket 
      residues on catalysis.
PG  - 12028-37
AB  - 3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion 
      between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 
      3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the 
      rate-determining step in this physiologically important reaction, rat liver 
      3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in 
      fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. 
      Using single and multiple turnover experiments to monitor the NADPH-dependent 
      reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were 
      identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic 
      isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 
      at pL 6.0, suggest that the slow chemical transformation is significantly 
      rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, 
      single and multiple turnover experiments showed a k(lim) and burst kinetics 
      consistent with product release as being rate-limiting overall. When NAD(+) was 
      substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was 
      identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT 
      plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 
      forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight 
      binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 
      38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics 
      observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the 
      R276M mutant confirms that the release of the NADPH product is the slow event; 
      and in its absence, chemistry becomes rate-limiting. W227 is a critical 
      hydrophobic residue at the steroid binding site, and when it is mutated to 
      alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase 
      kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also 
      abolished. In the W227A mutant, the slow release of NADPH is no longer observed 
      since the chemical transformation is now even slower. Thus, residues in the 
      cofactor and steroid-binding site can alter the rate-determining step in the 
      NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting 
      overall.
FAU - Heredia, Vladi V
AU  - Heredia VV
AD  - Department of Biochemistry & Biophysics, University of Pennsylvania School of 
      Medicine, Philadelphia, Pennsylvania 19104-6084, USA.
FAU - Penning, Trevor M
AU  - Penning TM
LA  - eng
GR  - DK47015/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Isotopes)
RN  - 0 (Solvents)
RN  - 08J2K08A3Y (Dihydrotestosterone)
RN  - 25126-76-5 (Androstane-3,17-diol)
RN  - 53-59-8 (NADP)
RN  - EC 1.1.1.50 (3-alpha-Hydroxysteroid Dehydrogenase (B-Specific))
SB  - IM
MH  - 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)/*chemistry/genetics/*metabolism
MH  - Androstane-3,17-diol/chemistry/*metabolism
MH  - Animals
MH  - Binding Sites
MH  - Catalysis
MH  - Dihydrotestosterone/chemistry/*metabolism
MH  - Isotopes
MH  - Kinetics
MH  - Molecular Structure
MH  - Mutation/*genetics
MH  - NADP/*metabolism
MH  - Oxidation-Reduction
MH  - Rats
MH  - Solvents/chemistry
EDAT- 2004/09/24 05:00
MHDA- 2004/11/13 09:00
CRDT- 2004/09/24 05:00
PHST- 2004/09/24 05:00 [pubmed]
PHST- 2004/11/13 09:00 [medline]
PHST- 2004/09/24 05:00 [entrez]
AID - 10.1021/bi0489762 [doi]
PST - ppublish
SO  - Biochemistry. 2004 Sep 28;43(38):12028-37. doi: 10.1021/bi0489762.