ELECTROPHILIC ASSISTANCE BY ASP-99 OF 3-OXO-DELTA-5-STEROID ISOMERASE

Citation
Ld. Thornburg et al., ELECTROPHILIC ASSISTANCE BY ASP-99 OF 3-OXO-DELTA-5-STEROID ISOMERASE, Biochemistry, 37(29), 1998, pp. 10499-10506
Citations number
55
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
37
Issue
29
Year of publication
1998
Pages
10499 - 10506
Database
ISI
SICI code
0006-2960(1998)37:29<10499:EABAO3>2.0.ZU;2-1
Abstract
3-Oxo-Delta(5)-steroid isomerase (Delta(5)-3-ketosteroid isomerase, KS I; EC 5.3.3.1) catalyzes the conversion of a Variety of beta,gamma-uns aturated 3-oxosteroids to their corresponding alpha,beta-unsaturated i somers at rates that approach the diffusion limit for specific substra tes. The reaction proceeds through a dienolate intermediate, with two amino acid residues (Asp-38 and Tyr-14) known to be involved in cataly sis. When the complete three-dimensional structure of KSI was determin ed recently by NMR methods, an additional polar residue (Asp-99) was f ound in the active site and this group was shown to be important for c atalytic activity. In this work, we examine the properties of several mutant KSIs to determine the nature of catalysis by Asp-99 of KSI. The electrophoretic mobilities of wild-type (WT) KSI and several mutants (D99A, D99N, D38N, and D38N/D99A) on native gels were determined at pH values ranging from 6.0 to 8.5. The results demonstrate that the pK(a ), of Asp-99 is > 8.5 in wild-type KSI.: The pH-rate profiles for the D99A, D99N, and D38H/D99A mutants of KSI were also determined. For all three mutants, k(cat) and k(cat)/K-M do not decrease at high pH, in c ontrast to those for WT and D38H, which lose activity above pH 9 and 8 , respectively. Mutation of Asp-99 to Asn decreases k(cat) for the sub strate 5-androstene-3,17-dione by 27-fold and k(cat)/K-M by 23-fold, s ubstantially less than the loss of activity (3000-fold in k(cat) and 2 200-fold in k(cat)/K-M) observed when Asp-99 is mutated to Ala, consis tent with a hydrogen bonding role for Asp-99. Taken together, these re sults provide evidence that Asp-99 participates in catalysis in its pr otonated form, with a pK(a), of >9 in WT and similar to 8.5 in the D38 H mutant. Asp-99 likely donates a hydrogen bond to O-3 of the steroid, helping to stabilize the transition state(s) of the KSI-catalyzed rea ction.