Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli

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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 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.