Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme.
GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.
Hatakeyama
K
K
Department of Medical Chemistry, Osaka Medical College, Japan.
Harada
T
T
Suzuki
S
S
Watanabe
Y
Y
Kagamiyama
H
H
D000619:Aminohydrolases / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006136:GTP Cyclohydrolase / Q000302:isolation & purification* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006882:Hydroxyapatites; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains
D006882:Hydroxyapatites; D007202:Indicators and Reagents; D006160:Guanosine Triphosphate; D017886:Durapatite; D000619:Aminohydrolases; D006136:GTP Cyclohydrolase