guanosine-triphosphate and phosphohistidine

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

Topics: Animals; Binding Sites; Biocatalysis; Crystallization; Crystallography, X-Ray; Glutamic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Histidine; Magnesium; Models, Molecular; Protein Conformation; Succinate-CoA Ligases; Succinic Acid; Swine

Although [Cl(-)](i) regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe(3+)-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins. Phosphate on [(32)P]annexin I, phosphorylated using both [gamma-(32)P]ATP and [gamma-(32)P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Annexin A1; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Epithelium; Guanosine Triphosphate; Histidine; Molecular Sequence Data; Molecular Weight; Phosphoamino Acids; Phosphorylation; Respiratory System; Sheep; Trachea

The chemical nature of the enzyme-nucleotide phosphoramidate reaction intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets of Artemia franciscana cysts to synthesize diguanosine tetraphosphate (Gp4G) has been investigated. Labeling of the enzyme with [alpha-32P]GTP followed by isolation of the labeled phosphoamino acid by periodate treatment and alkaline hydrolysis and comparison of the product with phosphoamino acid standards by thin-layer and ion-exchange chromatography showed that the linkage involves the Nepsilon2 ring nitrogen of an enzyme histidyl residue. Thus, this enzyme is distinct from the mRNA capping enzymes which can also synthesize Gp4G but which employ a lysyl-nucleotide intermediate. Based on its reaction mechanism and substrate specificity, GTP:GTP guanylyltransferase may belong to the GAFH superfamily which includes the histidine triad proteins, Ap4A phosphorylases, and galactose-1-phosphate uridylyltransferase.

Topics: Animals; Artemia; Dinucleoside Phosphates; Guanosine Triphosphate; Histidine; Multienzyme Complexes; Nucleotidyltransferases; Phosphoric Monoester Hydrolases

One major substrate protein was phosphorylated with [gamma-32P]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigrated with beta-subunits of heterotrimeric GTP-binding proteins (G proteins) in different gel systems. Upon solubilization of the phosphorylated membranes, the phosphoprotein could be immunoprecipitated by a G protein beta-subunit-specific antiserum. The beta-subunit phosphorylation was transient and was found to be specific for GTP and its analog, guanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was specifically removed by GDP, suggesting that the phosphate can be retransferred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were required for both phosphorylation and dephosphorylation. The phosphorylation was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membranes with the histidine-modifying agent, diethyl pyrocarbonate, resulted in a loss in phosphate incorporation. The data suggest that G protein beta-subunits are involved in a guanine nucleotide-specific enzymatic activity transferring the gamma-phosphate from GTP to GDP, presumably at G protein alpha-subunits, via a phosphohistidine intermediate.

Topics: Biological Transport; Cell Membrane; Diethyl Pyrocarbonate; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Histidine; Humans; Hydroxylamine; Hydroxylamines; Kinetics; Leukemia, Promyelocytic, Acute; Macromolecular Substances; Magnesium; Manganese; Membrane Proteins; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Tumor Cells, Cultured