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E M Westbrook

Publications and source records attributed to E M Westbrook.

24 records · Page 2Linked to original sources
PubMed1984

The 6-A crystal structure of delta 5-3-ketosteroid isomerase. Architecture and location of the active center.

The crystal structure of the dimeric steroid metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1), has been solved to 6-A resolution by multiple isomorphous replacement, augmented by real space direct methods. The unit cell is hexagonal (space group P6122) with dimensions a = b = 65.4 A, c = 504 A, and contains four identical 13,400-dalton protomers in each of its 12 asymmetric units. The 504-A c axis required double focusing mirrors (Franks optics) to resolve the reflections. The complexity of the combined local and lattice symmetry necessitated direct methods to establish the positions of heavy atoms in even the simplest of the isomorphous derivatives. The electron density map clearly showed both (a) the elaborate packing scheme of protomers, which accounts for this large and complicated unit cell, and (b) the coarse features of the functional dimer. The steroid-binding site has been established by imaging the bound inhibitor, 4-acetoxymercuriestradiol, in a difference Fourier map. Each of the dimer's two steroid-binding sites lies completely within one subunit but close enough to the opposing subunit that functional interactions may be possible.

Binding Sites↗
PubMed1984

Isolation and crystallization of unadenylylated glutamine synthetase from Salmonella typhimurium.

The enzyme glutamine synthetase (GS) has been isolated from a mutant strain of Salmonella typhimurium, constructed by Kustu, which lacks the enzymatic activity for adenylylation of glutamine synthetase. Thus the purified GS is uniformly unadenylylated, as confirmed by gel electrophoresis and enzyme assays. It crystallizes readily in many morphologies, at least six of which are distinct polymorphs. The most favorable crystal form for structural studies belongs to space group C2, with unit cell dimensions a = 235.5 A, b = 134.5 A, c = 200.1 A, beta = 102.8 degrees, and with one GS molecule per asymmetric unit. The crystals diffract to about 2.8 A resolution in rotation X-ray photographs and thus appear suitable for structural studies at moderate resolution. These crystals are isomorphous with crystalline GS from Escherichia coli in both adenylylated and unadenylylated states, suggesting that the enzymes from the two bacteria are similar molecules, and that adenylylation does not greatly affect the conformation of the molecule.

Adenosine Monophosphate↗
PubMed1982

X-ray grade crystals of diphtheria toxin.

Diphtheria toxin, complexed with the endogenous dinucleotide ApUp, has been crystallized under novel conditions involving high concentrations of both polyethylene glycol and salt. Among four crystal forms obtained, one was found suitable for high resolution structural analysis by x-ray diffraction. This form is triclinic (space group P1) with 2 molecules per unit cell; unit cell parameters are a = 70.8 A, b = 70.7 A, c = 65.3 A, alpha = 95.2 degrees, beta = 91.3 degrees, gamma = 99.7 degrees.

Crystallization↗
PubMed1980

On the number of steroid-binding sites of delta 5-3-oxosteroid isomerase.

The number of steroid-binding sites in delta 5-3-oxosteroid isomerase of Pseudomonas testosteroni (EC 5.3.3.1) has been determined from measurements of the red shift of the ultraviolet chromophore of 19-nortestosterone upon binding to the enzyme. The experiments include spectroscopic measurements when limiting concentrations of either 19-nortestosterone or isomerase are titrated with varying concentrations of the complementary ligand. Analysis of the results indicates one binding site per subunit (Mr 13 394). Scatchard plots indicate a single family of equivalent binding sites. 5, 10-Secoestr-5-yne-3, 10, 17-trione is a suicide substrate of isomerase [Batzold & Robinson (1975) J. Am. Chem. Soc. 97, 2576]. The time course for inactivation of isomerase with an excess of 5, 10-seco[7-3H]estr-5-yne-3, 10, 17-trione was parallel to the covalent incorporation of steroid and gave a final stoichiometry of nearly one steroid molecule per subunit of enzyme. Alkylation of [14C] isomerase with excess of this 3H-labeled steroid followed by gel-filtration and dialysis gave an inactivated enzyme with a 3H/14C ratio that corresponds to one molecule of steroid bound per subunit; this stoichiometry was constant over a wide range of protein concentrations (0.1--10 mg/ml). Diffusion of [3H]progesterone into hexagonal crystals of isomerase showed that at saturation one steroid molecule was bound per protomer. Taken together these findings strongly support the conclusion that one molecule of steroid is bound per subunit of isomerase both in solution and in the crystal state.

Binding Sites↗