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.