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Site-directed mutagenesis of the active site cysteine in Klebsiella aerogenes urease.

Open AccessPublished:October 05, 1992DOI:https://doi.org/10.1016/S0021-9258(19)88659-3
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      Cysteine 319 in the large subunit of Klebsiella aerogenes urease was identified as an essential catalytic residue based on chemical modification studies (Todd, M.J., and Hausinger, R.P. (1991) J. Biol. Chem. 266, 24327-24331). Through site-directed mutagenesis, this cysteine has been changed independently to alanine, serine, aspartate, and tyrosine. None of these mutations (C319A, C319S, C319D, and C319Y, respectively) affected the size or level of synthesis of the urease subunits as monitored by polyacrylamide gel electrophoresis. The wild type enzyme and each of the mutant proteins was purified and their properties were compared. The C319Y protein possessed no detectable activity, while activity was reduced in C319A, C319S, and C319D to 48, 4.5, and 0.03% of wild type levels under normal assay conditions. All of the active mutants had a small increase in Km when compared to the wild type value. The active mutants displayed a greatly reduced sensitivity to inactivation by iodoacetamide in comparison to the wild type enzyme, confirming our previous assignment of the essential cysteine to this residue based on active site peptide mapping. In contrast to the wild type enzyme, inactivation of the mutant proteins was not affected by the presence of the competitive inhibitor phosphate, suggesting that the remaining slow rate of iodoacetamide inactivation is due to modification away from the active site. The pH dependence of urease activity was substantially altered in the active mutants with C319S and C319D showing a pH optimum near 5.2, and C319A near 6.7, compared to the pH 7.75 optimum of wild type urease. These data are consistent with Cys-319 facilitating catalysis at neutral and basic pH values by participating as a general acid.

      REFERENCES

        • Andrews R.K.
        • Reithel F.J.
        Arch. Biochem. Biophys. 1970; 141: 538-546
        • Dixon N.E.
        • Gazzola C.
        • Blakeley R.L.
        • Zerner B.
        J. Am. Chem. Soc. 1975; 87: 4131-4133
        • Dixon N.E.
        • Riddles P.W.
        • Gazzola C.
        • Blakeley R.L.
        • Zerner B.
        Can. J. Biochem. 1980; 58: 1335-1344
        • Kunkel T.A.
        • Roberts J.D.
        • Zakour R.A.
        Methods Enzymol. 1987; 154: 367-382
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Lee M.H.
        • Mulrooney S.B.
        • Renner M.J.
        • Markowicz Y.
        • Hausinger R.P.
        J. Bacteriol. 1992; 174: 4324-4330
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        J. Biol. Chem. 1951; 193: 265-275
        • Mobley H.L.T.
        • Hausinger R.P.
        Microbiol. Rev. 1989; 53: 85-108
        • Mulrooney S.B.
        • Hausinger R.P.
        J. Bacteriol. 1990; 172: 5837-5843
        • Mulrooney S.B.
        • Pankratz H.S.
        • Hausinger R.P.
        J. Gen. Microbiol. 1989; 135: 1769-1776
        • Nakano H.
        • Takenishi S.
        • Watanabe Y.
        Agric. Biol. Chem. 1984; 48: 1495-1502
        • Norris R.
        • Brocklehurst K.
        Biochem. J. 1976; 159: 245-257
        • Saada A.-B.
        • Kahane I.
        Zentrabl. Bakteriol. Parisitenkd. Infektionskr. Hyg. Abt. I Orig. Reihe A. 1988; 269: 160-167
        • Sanger F.
        • Nicklen S.
        • Coulson A.R.
        Proc. Natl. Acad. Sci. U. S. A. 1977; 80: 3963-3965
        • Takishima K.
        • Suga T.
        • Mamiya G.
        Eur. J. Biochem. 1988; 175: 151-165
        • Todd M.J.
        • Hausinger R.P.
        J. Biol. Chem. 1987; 262: 5963-5967
        • Todd M.J.
        • Hausinger R.P.
        J. Biol. Chem. 1989; 264: 15835-15842
        • Todd M.J.
        • Hausinger R.P.
        J. Biol. Chem. 1991; 266: 10260-10267
        • Todd M.J.
        • Hausinger R.P.
        J. Biol. Chem. 1991; 266: 24327-24331
        • Weatherburn M.W.
        Anal. Chem. 1967; 39: 971-974
        • Winkler R.G.
        • Blevins D.G.
        • Polacco J.C.
        • Randall D.D.
        Trends Biochem. Sci. 1988; 13: 97-100
        • Zerner B.
        Bioorgan. Chem. 1991; 19: 116-131