Isolation and characterization of human liver guanine deaminase

Abstract

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolate [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 p I 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 K m for guanine was determined to be 1.53 × 10 −5 m at pH 6.0 and 2 × 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 K i of 1.53 × 10 −5 m and a K i of 5 × 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 × 10 −4 m and a 24% loss at 1 × 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 × 10 −4 m. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 °C, with a loss of almost all activity at 65 °C with 30 min incubation. Two p K a 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.