Reaction energetics of a mutant 3-oxo-delta 5-steroid isomerase with an altered active site base (D38E).
3-Oxo-delta 5-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated delta 4-isomers through the formation of an intermediate dienol. Mutation of the catalytic base (Asp-38) to Glu (D38E) has been found to reduce kcat/Km for the isomerization of 5-androstene-3,-17-dione (1) to 4-androstene-3,17-dione (3) by about 300-fold (Zawrotny et al., 1991). The free energy profile for the D38E enzyme was determined from a combination of steady state kinetics and stopped-flow kinetics with the independently generated dienol intermediate (2). A comparison of the energetics of D38E with that of the wild type enzyme (WT) shows that the only significant difference is a reduction in the rates of the chemical steps for the interconversion of 1, 2, and 3 on the enzyme surface by about 10(3)-fold for D38E. The relative energy levels for all bound species are nearly identical for WT and D38E, whereas the transition states for both enolization and ketonization are destabilized by 3-4 kcal/mol. The effect of the D38E mutation on the energetics of KSI is comparable to the corresponding effect of the E165D mutation on the energetics of triosephosphate isomerase (TIM).
Zawrotny
M E
ME
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228-5398.
Pollack
R M
RM
D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D009154:Mutation; D013251:Steroid Isomerases / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics
D001224:Aspartic Acid; D018698:Glutamic Acid; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D000432:Methanol