Purification and Characterization of Rat Liver GTP Cyclohydrolase I

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 PM. The V,,, value of the enzyme was 45 nmollmin. mg protein. The GTP concentration producing half-maximal velocity was 30 I.LM at a KC1 concentration of 0.1 M. This value increased as the KC1 concentration rose, without any change in V,,, or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.