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1.
J Biol Chem ; 289(51): 35605-19, 2014 Dec 19.
Article in English | MEDLINE | ID: mdl-25378390

ABSTRACT

Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases.


Subject(s)
Allosteric Regulation , Enzyme Inhibitors/chemistry , Metalloendopeptidases/chemistry , Protein Structure, Tertiary , Allosteric Site , Animals , Binding Sites/genetics , Biocatalysis/drug effects , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fluorescence Polarization , Kinetics , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Models, Chemical , Models, Molecular , Molecular Structure , Mutation, Missense , Protein Binding , Rats , Substrate Specificity
2.
Environ Sci Pollut Res Int ; 21(10): 6400-9, 2014 May.
Article in English | MEDLINE | ID: mdl-23532536

ABSTRACT

Copper (Cu) metabolism is altered in rats fed diets high in molybdenum (Mo) and low in Cu. This 10-week study was carried out to examine the effects of supplemental Mo (7.5-240 µg/g diet) on male Sprague-Dawley rats fed diets adequate in Cu (5 µg/g diet) and to determine the susceptibility of Mo-treated animals to the environmental pollutant 3,3',4,4'-tetrabromobiphenyl (TBB). After 7 weeks of dietary treatment, half of the rats in each group received a single IP injection of TBB (150 µM/kg bw), while the other half received the corn oil vehicle. Rats sacrificed at 10 weeks showed no effects of Mo on growth, feed efficiency, or selected organ or tissue weights. Dose-dependent effects on plasma Mo (0-5.1 µg/mL), plasma Cu (0.95-0.20 µg/mL), and bone Cu (3.4-10 µg/g) in control through the high dose were found. Cu sequestration in the bone of Mo-treated rats is a new finding. TBB treatment resulted in dramatic weight loss and loss of absolute organ mass. Relative organ weights were increased, except for the thymus. TBB altered the concentrations of certain amino acids. Compared to control rats, this polybrominated biphenyl congener significantly decreased plasma Cu and ceruloplasmin at higher concentrations of dietary Mo and promoted the process of plasma Cu decrease by Mo, suggesting a combined effect.


Subject(s)
Copper/toxicity , Hazardous Substances/toxicity , Molybdenum/toxicity , Polybrominated Biphenyls/toxicity , Animals , Ceruloplasmin/metabolism , Copper/metabolism , Diet , Dietary Supplements , Hazardous Substances/administration & dosage , Hazardous Substances/metabolism , Injections, Intraperitoneal , Male , Molybdenum/metabolism , Organ Size/drug effects , Polybrominated Biphenyls/administration & dosage , Polybrominated Biphenyls/metabolism , Rats , Rats, Sprague-Dawley
3.
Biochem J ; 413(3): 417-27, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18412546

ABSTRACT

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.


Subject(s)
Agriculture/methods , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Biotechnology/methods , Amidohydrolases/genetics , Amino Acid Sequence , Arabidopsis/enzymology , Arabidopsis/genetics , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Substrate Specificity , Tyrosine/metabolism
4.
J Biol Chem ; 282(13): 9722-9732, 2007 Mar 30.
Article in English | MEDLINE | ID: mdl-17251185

ABSTRACT

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.


Subject(s)
Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Amino Acid Substitution/genetics , Crystallography, X-Ray , Humans , Metalloendopeptidases/genetics , Substrate Specificity/genetics
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