Characterization of purified guanine aminohydrolase.

Guanine aminohydrolase (GAH) (E.C. 3.5.4.3) was purified by affinity chromatography on 9-(p-β-aminoethoxyphenyl)guanine-Sepharose to a specific activity of 35.5 units/mg. The molecular weight of the enzyme was estimated to be 110,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) showed that the enzyme was composed of subunits with molecular weights of approximately 52,000. Data from SDS-gel electrophoresis in a discontinuous buffer system and from isoelectric focusing in the presence of 8-m urea indicated that more than one type of subunit were present. This was consistent with multiple forms of the native enzyme seen by electrophoresis and isoelectric focusing in polyacrylamide gels. The isoelectric points for the different forms of GAH were in the range of 4.65–4.85. Amino acid analyses showed cysteine to be the minimum amino acid and gave a calculated molecular weight for GAH of 53,016 when the assumption that there were four cysteines per subunit was made. Guanine, 8-azaguanine, and 6-thioguanine served as substrates for the enzyme but 3-deazaguanine, a potent competitive inhibitor of GAH, did not. Fluoride ion inhibited the enzyme in a noncompetitive manner, and this inhibition decreased as pH increased. Variation of the kinetic parameters with pH suggested that hydroxide ion might be the second substrate and that a functional group on the enzyme with a pK_a near 5.6 was involved in the reaction. The enzyme was inactivated by treatment with p-hydroxymercurobenzoate and by photooxidation in the presence of rose bengal. Two plausible mechanisms are proposed for the reaction catalyzed by GAH.