ABSTRACT
Dehydroepiandrosterone sulfotransferase converts dehydroepiandrosterone (DHEA) and some other steroids to their sulfonated forms. The human enzyme has been crystallized in the presence of substrate (DHEA) alone, in the presence of substrate and non-sulfated cofactor analogue (PAP) and in the absence of both substrate and PAP in our laboratory, with data sets collected at a synchrotron source. The crystals of the uncomplexed form belong to the orthorhombic space group C222(1), with unit-cell parameters a = 85.26, b = 87.69, c = 108.20 A and data 99.2% complete to 2.35 A resolution. The DHEA complex crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.46, b = 127.49, c = 44.59 A and data 92.9% complete to 2.15 A resolution. The ternary complex crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.25, b = 87.28, c = 138.86 A and data 98.6% complete to 2.50 A resolution. Preliminary molecular-replacement solutions indicate significant variations in dimer formation.
Subject(s)
Apoenzymes/chemistry , Sulfotransferases/chemistry , Crystallization , Crystallography, X-Ray , Dehydroepiandrosterone/chemistry , Humans , Models, Molecular , Protein ConformationABSTRACT
The enzyme human muscle fructose-1,6-bisphosphatase, which plays a critical role in gluconeogenesis, has been crystallized in the presence of 2-propanol, polyethylene glycol and magnesium chloride at pH 7.5. The space group was determined to be P4(2)2(1)2, with unit-cell parameters a = b = 73.57, c = 146.50 A, alpha = beta = lambda = 90 degrees and one subunit in the asymmetric unit. A 99.6% complete data set to 2.04 A has been collected at the National Synchrotron Light Source.
Subject(s)
Fructose-Bisphosphatase/chemistry , Muscles/enzymology , 2-Propanol/chemistry , Crystallization , Crystallography, X-Ray , Humans , Magnesium/chemistry , Polyethylene Glycols/chemistry , Protein ConformationABSTRACT
In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the androgen receptor (AR) activity by catalyzing the inactivation of 5 alpha-dihydrotestosterone (the most natural potent androgen) to 5 alpha-androstane-3 alpha,17 beta-diol. In this report, the crystallization of a human prostatic type 3 3 alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily, is described. Two different crystal forms of the complex between the human type 3 3 alpha-HSD, NADP(+) and testosterone have been obtained using PEG as precipitant. Crystal form I, which diffracts to 1.6 A, belongs to the monoclinic space group P2(1), with unit-cell parameters a = 55.07, b = 87.15, c = 76.88 A, beta = 107.37 degrees and two subunits in the asymmetric unit. A complete data set has been collected at 1.8 A. Crystal form II, which diffracts to 2.6 A, belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 143.59, c = 205.86 A, alpha = beta = 90, gamma = 120 degrees and two subunits in the asymmetric unit.
Subject(s)
3-Hydroxysteroid Dehydrogenases/chemistry , Prostate/enzymology , 3-Hydroxysteroid Dehydrogenases/classification , 3-Hydroxysteroid Dehydrogenases/metabolism , Binding Sites , Crystallization , Humans , Male , Protein Subunits , Substrate Specificity , Testosterone/metabolism , X-Ray DiffractionABSTRACT
The snake muscle fructose 1,6-bisphosphatase, a typical allosteric enzyme which plays important roles in gluconeogenesis, was crystallized in the presence of polyethylene glycol 3350 and magnesium chloride at pH 8.5. The crystals diffract to 2.3 A on a rotating-anode X-ray source. The space group was determined to be either P3121 or its enantiomorph P3221, with unit-cell parameters a = b = 83.7, c = 202.41 A, alpha = beta = 90 and gamma = 120 degrees. There are two subunits in the asymmetric unit. Preliminary molecular-replacement studies indicate that the first enantiomorph is the correct one.
Subject(s)
Fructose-Bisphosphatase/chemistry , Muscles/enzymology , Animals , Crystallization , Crystallography, X-Ray , Protein Conformation , SnakesSubject(s)
Estradiol/chemistry , Hydroxysteroid Dehydrogenases/chemistry , Binding Sites , Breast Neoplasms/enzymology , Carcinoma/enzymology , Catalysis , Crystallography , Estradiol/metabolism , Estrone/metabolism , Female , Gonads/metabolism , Humans , Hydroxysteroid Dehydrogenases/metabolism , Models, Molecular , Protein Conformation , Substrate SpecificityABSTRACT
The crystal structure of a complex between a bivalent peptidyl pyridinium methyl ketone inhibitor and human alpha-thrombin has been solved and refined at 2.0 A to an R factor of 0.18. The inhibitor, (D)cyclohexylalanine-Pro-Arg-(CH2N+C5H4CH2CO)-(Gly)4-Asp- Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-cyclo-hexylalanine-(D)Glu (coded P596), which forms a reversible covalent complex with thrombin, is highly potent with a Ki = 4.6 +/- 1.0 x 10(-14) M, lower than that of recombinant hirudin. The N-terminal, active-site-directed portion of the inhibitor is linked to the fibrinogen recognition exosite binding portion by a tetraglycine segment. The strong electron-withdrawing effect provided by the permanent positive charge on the pyridinium nitrogen makes the arginyl carbonyl carbon more susceptible to nucleophilic attack. In the crystal, a covalent P596-thrombin complex is observed. The electron density surrounding the active site portion and the pyridinium of the inhibitor is very well defined, clearly showing the existence of a covalent bond between the Ser195 O gamma and the now tetrahedral carbon of the inhibitor. The decreased binding ability of thrombin inhibitors containing N-terminal acetylation is discussed as is the effect of replacing the P3 (D)phenylalanine with (D)cyclohexylalanine. The electron density surrounding the remainder of the inhibitor is generally well defined, the exceptions being the C-terminal (D)Glu, the highly flexible tetraglycine linker, and some of the solvent-directed side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Antithrombins/chemistry , Peptides/chemistry , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Anions , Binding Sites , Crystallography, X-Ray , Humans , Molecular Sequence Data , Molecular Structure , Oxygen/chemistry , Thrombin/chemistryABSTRACT
Two linear synthetic peptides, N-tBoc-Pro-Gly-Ala-NHCH3 and N-tBoc-Pro-D-Ala-Ala-NHCH3, have previously been shown by us to complex with Ca2+ and form 2:1 (peptide:calcium) complexes. Here we report their binding to Pr3+ and demonstrate, by 1H NMR, the peptide-mediated transport of Pr3+ across dimyristoylphosphatidylcholine unilamellar vesicles via a 2:1 ion-sandwich complex.
Subject(s)
Oligopeptides , Praseodymium , Biological Transport , Calcium , Choline , Ionophores , Kinetics , Models, Biological , Protein Conformation , Structure-Activity RelationshipABSTRACT
A C-type lactate dehydrogenase isozyme has been purified to homogeneity from the liver of the Atlantic cod. The enzyme consists of four identical subunits each with a mol. wt of 35,000. Optimum concentrations, Kms, and relative activities were determined for various substrates along with the optimum pH for the reaction with pyruvate and lactate. Glyoxalate, alpha-ketobutyrate, alpha-ketovalerate, and alpha-ketoglutarate were significantly reduced but branched chain alpha-ketoacids were not utilised as substrates. Substrate inhibition was observed for both lactate and pyruvate as is generally found for B-type lactate dehydrogenase isozymes but the lactate optimum concentration and Km more closely resemble the A-type lactate dehydrogenases.
