Characteristics of guanosine triphosphate cyclohydrolase I purified from Escherichia coli

Abstract

GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of the pteridine portion of folic acid, was purified from Escherichia coli by 3,900-fold to apparent homogeneity. Its molecular weight is estimated at 210,000. At relatively high concentrations of salt (e.g. 0.3 M KCl) the enzyme can be dissociated into seemingly identical subunits of 51,000 molecular weight. Removal of the salt allows reassociation. GTP, ATP, and inorganic orthophosphate at concentration of 5 muM, 100muM, and 0.2 mM, respectively, promote the reassociation of the subunits even in the presence of 0.3 M salt. The subunits have little or no catalytic activity. When the enzyme was subjected to electrophoresis on polyacrylamide gel under denaturing conditions (in the presence of sodium dodecyl sulfate) only one protein band was evident; its molecular weight was estimated at 25,500. Proline was determined as the only NH2-terminal amino acid residue of the enzyme. These observations suggest that the enzyme consists of four identical subunits and that each subunit contains two identical polypeptide chains. Enough GTP was bound to the enzyme to suggest that each polypeptide contains one GTP binding site. The Km value for GTP IS 0.02 MuM. ATP, dGTP, and guanosine 5'-tetraphosphate are competitive inhibitors with Ki values of 0.25 muM, 0.24 muM, and 0.13 muM, respectively. Orthophosphate is an uncompetitive inhibitor. The enzyme is relatively heat-stable; its half-life at 82 degrees is 7 min. Salt (NaCl, KCl, NH4Cl) at a concentration of 0.1 M activates the enzyme by 4- to 5-fold. The only products of the action of the enzyme are formate and the triphosphoester of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (H2-neopterin-PPP). The evidence strongly suggests that this single enzyme catalyzes 4 independent chemical reactions in the conversion of GTP to H2-neopterin-PPP.