ABSTRACT
The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet. Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.
Subject(s)
Candida/chemistry , DNA-Binding Proteins/chemistry , Protein Structure, Secondary , Transcription Factors/chemistry , Zinc/chemistry , Amino Acid Sequence , Copper/pharmacology , Cysteine/chemistry , Fungal Proteins , Molecular Sequence Data , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.
Subject(s)
Protein Conformation , Steroid Isomerases/chemistry , Amino Acid Sequence , Androstenedione/metabolism , Binding Sites , Dimerization , Estradiol/metabolism , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary , Solutions , Steroid Isomerases/genetics , Steroid Isomerases/metabolismABSTRACT
The stereochemistry of proton transfer in the isomerization of [4 beta-2H]-5-androstene-3,17- dione (1d) to 4-androstene-3,17-dione (3) catalyzed by 3-oxo-delta 5-steroid isomerase (KSI) has been reinvestigated. In H2O, approximately 65% of the label is retained in the product (3); of this, one-third is at C-4 and two-thirds at C-6 beta. When the same reaction is catalyzed by the D38E mutant of KSI, ca. 60% of the label is retained in the product, but almost all of it is at C-4. These reactions run in deuterium oxide result in 13% incorporation of a second deuterium with the wild type (WT) enzyme and 75% incorporation with the D38E mutant. When unlabeled 1 is isomerized in D2O, there is little incorporation of deuterium with WT (ca. 5 at. %) but substantial incorporation with D38E (130 at. %). These results are consistent with competitive abstraction of both the C-4 alpha and C-4 beta protons, as proposed by Viger et al. [(1981) J. Am. Chem. Soc. 103, 4151], and demonstrate that the KSI reaction is not completely stereospecific. A mechanism is proposed to account for these observations.
Subject(s)
Androstenedione/chemistry , Androstenedione/metabolism , Steroid Isomerases/genetics , Steroid Isomerases/metabolism , Deuterium , Magnetic Resonance Spectroscopy , Mass Spectrometry , Point Mutation , Protons , StereoisomerismABSTRACT
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).
