43] GTP cyclohydrolase II from Escherichia coli

Abstract

This chapter discusses the guanosine triphosphate (GTP) cyclohydrolase II from Escherichia coli . E. coli contains two enzymes that catalyze the removal of carbon 8 of GTP as formate. One of these enzymes called “GTP cyclohydrolase I” catalyzes the first reaction in the pathway for the biosynthesis of the pteridine portion of folic acid. The second enzyme called “GTP cyclohydrolase II” can be distinguished from the first enzyme by its properties and the products of its action on GTP. These products are formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine 5'-phosphate. The activity of GTP cyclohydrolase II can be assayed by the measurement of the amount of radioactive formate produced from [8- 14 C]GTP. This is most conveniently determined by the treatment of an incubated reaction mixture with activated charcoal. The unreacted radioactive GTP is adsorbed to the charcoal, whereas the radioactive product, formic acid, is not adsorbed. GTP cyclohydrolase II can be stored at pH 8.0 without appreciable loss of activity at –20° if the protein concentration is 1 mg/ml or higher. The presence of ethylenediaminetetraacetic acid (EDTA) and an excess of MgC1 2 (over EDTA) also helps stabilize activity. The enzyme rapidly loses activity at temperatures of 60° or higher.