Rabbit Liver Guanine Deaminase

Homogeneous guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) is a single polypeptide chain of mean molecular weight of 55,000, as determined by Sephadex gel filtration and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Irreversible inactivation was observed upon incubation with p-chloromercuribenzoate, at a concentration of 1.0 x 10-4 m. Iodoacetic acid and iodoacetamide were mildly inhibitory at a concentration of 2.0 x 10-3 m. The purine precursor 5-aminoimidazole-4-carboxamide was a potent competitive inhibitor of the enzyme, with a Ki of 3.05 x 10-5 m. Its ribonucleoside derivative (5-aminoimidazole-4-carboxamide ribonucleoside) and tetrahydrofolic acid were also observed to be competitive inhibitors of the enzyme. Their respective Ki values were 2.58 x 10-4 m for tetrahydrofolic acid and 5.68 x 10-4 m for 5-aminoimidazole-4-carboxamide ribonucleoside. A single pH optimum at pH 6.8 was observed in Tris, citrate, and phosphate buffers. The Km values determined for substrates of the enzyme, in 0.2 m phosphate buffer at 40°, were 1.25 x 10-5 m for guanine at pH 7.0, 3.33 x 10-4 m for 8-azaguanine at pH 6.0, and 8.0 x 10-4 m for 6-thioguanine at pH 7.0. At saturating substrate concentration, deuterium isotope was observed to enhance the catalytic rate of the enzyme with a Kd2o/Kh2o of 1.58. Deuterium isotope effect on proton transfer was observed at nonsaturating substrate concentration, and a Kh2o/Kd2o of 1.46 was observed at 20% D2O. The enzyme was labile to heat, and a 42% loss of enzyme activity was sustained upon incubation at 55° for 25 min. Irreversible and almost total denaturation was observed upon incubation at 60° for 25 min. The activation energy was linear between 20° and 50°, and an activation energy of 4.94 Cal per mole was calculated for the enzyme. The experimental evidence strongly implicated histidine and cysteine —SH as the catalytic species at the active site of the enzyme.