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 μM) and UTP-agarose (110 μM). 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 μM 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.