ABSTRACT
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
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.
