Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli

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 Escherichiu 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 1 has a relatively high apparent K,. The values are slightly different for enzyme purified by GTP-agarose (100 pM) and UTP-agarose (1 10 pM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in V,,, 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 pM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobioptcrin. 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.CTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. This is the first step in the multienzyme biosynthesis of the cofactor, tetrahydrobiopterin, of the aromatic amino acid hydroxylases [l]. GTP cyclohydrolase I has been described in many biological systems (for review see [2]

Biosynthesis of Methanopterin in Methanobacterium thermoautotrophicum

“…Interestingly, activity of GTP cyclohydrolase I in crude extract of M thennoautotrophicum has only been detected when divalent cations (Mg2+ or Mn2+) were present. This is in contrast to GTP cyclohydrolase I from E. coli which does not require divalent cations and is active in the presence of EDTA, although an activating effect of divalent cations has been described recently (28).…”

Discovery of a New Prokaryotic Type I GTP Cyclohydrolase Family

“…It is encoded in Escherichia coli by the folE gene (8,9) and catalyzes a complex reaction (10) that begins with hydrolytic ring opening of the purine ring at C-8 to generate an N-formyl intermediate, which is then the site for a second hydrolysis with concomitant loss of C-8 as formic acid. In the subsequent steps of the reaction, the ribosyl moiety undergoes ring opening and an Amadori rearrangement followed by cyclization to generate the pterin ring in THF (Fig.…”

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

“…The first step of pterin synthesis is of special interest, because it commits GTP to pterin production and is considered to control flux into the pathway (9,10). This step, mediated by GTP cyclohydrolase I (GCHI, EC 3.5.4.16), is a complex ring expansion that converts GTP to dihydroneopterin triphosphate and formate ( Fig.…”