Active site topology and reaction mechanism of GTP cyclohydrolase I.
GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.
Nar
H
H
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Huber
R
R
Auerbach
G
G
Fischer
M
M
Hösl
C
C
Ritz
H
H
Bracher
A
A
Meining
W
W
Eberhardt
S
S
Bacher
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D006136:GTP Cyclohydrolase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006136:GTP Cyclohydrolase