Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli.
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.
Schoedon
G
G
Department of Pediatrics, University of Zurich, Switzerland.
Redweik
U
U
Frank
G
G
Cotton
R G
RG
Blau
N
N
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014544:Uridine Triphosphate / Q000494:pharmacology
D002413:Cations, Divalent; D010446:Peptide Fragments; D001708:Biopterins; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D006136:GTP Cyclohydrolase; C003402:sapropterin; D008274:Magnesium; D002118:Calcium; D014544:Uridine Triphosphate