Isolation and characterization of human liver guanine deaminase.
Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.
D000619:Aminohydrolases / Q000302:isolation & purification*; D000620:Aminoimidazole Carboxamide / Q000494:pharmacology; D000645:Ammonium Sulfate; D001375:Azaguanine / Q000378:metabolism; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D006147:Guanine / Q000378:metabolism; D006148:Guanine Deaminase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006882:Hydroxyapatites; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity
D006882:Hydroxyapatites; D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D000620:Aminoimidazole Carboxamide; D006147:Guanine; D000619:Aminohydrolases; D006148:Guanine Deaminase; D001375:Azaguanine; D000645:Ammonium Sulfate