PMID:1459137
| Citation |
Schoedon, G, Redweik, U, Frank, G, Cotton, RG and Blau, N (1992) Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli. Eur. J. Biochem. 210:561-8 |
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| Abstract |
The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme. |
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| Keywords |
Allosteric Regulation; Amino Acid Sequence; Binding Sites; Biopterin/analogs & derivatives; Biopterin/metabolism; Biopterin/pharmacology; Calcium/pharmacology; Cations, Divalent; Chromatography, Affinity; Edetic Acid/pharmacology; Enzyme Activation/drug effects; Escherichia coli/enzymology; GTP Cyclohydrolase/antagonists & inhibitors; GTP Cyclohydrolase/chemistry; GTP Cyclohydrolase/metabolism; Guanosine Triphosphate/metabolism; Kinetics; Magnesium/pharmacology; Molecular Sequence Data; Peptide Fragments/chemistry; Sequence Homology, Amino Acid; Uridine Triphosphate/pharmacology |
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