Isolation and characterization of GTP cyclohydrolase I from mouse liver. Comparison of normal and the hph-1 mutant.
GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of pterin compounds, was purified from of C3H mouse liver by 192-fold to apparent homogeneity, using Ultrogel AcA34, DEAE-Trisacryl, and GTP-agarose gels. Its native molecular weight was estimated at 362,000. When the enzyme was subjected to electrophoresis on a denaturing polyacrylamide gel, only one protein band was evident, and its molecular weight was estimated at 55,700. The NH2-terminal amino acid of this enzyme was serine. These results indicate the enzyme consists of six to eight subunits. No coenzyme or metal ion was required for activity. This enzyme activity was inhibited by most of divalent cations and was slightly activated by potassium ion. The Km value for GTP was determined to be 17.3 microM. The temperature and pH optima for the activity were 60 degrees C and pH 8.0-8.5, respectively. The expected products, a dihydroneopterin compound and formic acid, were found in a molar ratio of 1.01. A polyclonal antiserum generated against the purified enzyme was used to compare GTP cyclohydrolase I from the hph-1 mutant and normal mouse. The hph-1 mutant liver contained only 8% of normal specific activity, but a normal amount of GTP cyclohydrolase I antigen as compared with the C3H mouse. Subunit molecular weight and electrophoretic behavior of GTP cyclohydrolase I from hph-1 mutant were not different from those of the enzyme from C3H mouse. These results suggest that the hph-1 mutation may involve alteration of the catalytic site but does not detectably alter the whole enzyme structure.
Cha
K W
KW
Department of Microbiology, Seoul National University, Korea.
Jacobson
K B
KB
Yim
J J
JJ
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006136:GTP Cyclohydrolase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology*; D051379:Mice; D009154:Mutation; D013050:Spectrometry, Fluorescence
D006136:GTP Cyclohydrolase