title: Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate.
abstract: GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.
authors.affiliation: Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Schramek
authors.forename: Andreas
authors.initials: A
authors.lastname: Bracher
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
authors.forename: Günter
authors.initials: G
authors.lastname: Auerbach
authors.forename: Herbert
authors.initials: H
authors.lastname: Nar
authors.forename: Robert
authors.initials: R
authors.lastname: Huber
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005561:Formates / Q000378:metabolism; D006136:GTP Cyclohydrolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D019798:Neopterin / Q000031:analogs & derivatives; D011621:Pteridines / Q000378:metabolism; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism
chemical_list: D005561:Formates; D011621:Pteridines; D011742:Pyrimidine Nucleotides; D012545:Schiff Bases; C030544:formic acid; C013490:dihydroneopterin triphosphate; D019798:Neopterin; D006160:Guanosine Triphosphate; D006136:GTP Cyclohydrolase