Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102

Journal of Molecular Biology
B StecE R Kantrowitz

Abstract

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes an...Continue Reading

References

Jan 1, 1992·Annual Review of Biophysics and Biomolecular Structure·J E Coleman
Mar 20, 1991·Journal of Molecular Biology·E E Kim, H W Wyckoff
Mar 1, 1991·Acta Crystallographica. Section A, Foundations of Crystallography·T A JonesM Kjeldgaard
Jun 25, 1985·Nucleic Acids Research·P CarterG Winter
Jun 28, 1988·Biochemistry·J E Butler-RansohoffE T Kaiser
Nov 20, 1985·Journal of Molecular Biology·J M SowadskiH W Wyckoff
Feb 1, 1996·Journal of Molecular Graphics·R KoradiK Wüthrich
Nov 7, 1996·Chemical Reviews·William N. Lipscomb, Norbert Sträter
Dec 15, 1961·Proceedings of the National Academy of Sciences of the United States of America·J H SCHWARTZ, F LIPMANN
Mar 9, 1964·Biochimica Et Biophysica Acta·J DAYAN, I B WILSON
Mar 1, 1994·Trends in Cell Biology·S M Brady-Kalnay, N K Tonks

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Citations

May 5, 2012·Purinergic Signalling·Herbert ZimmermannNorbert Sträter
Feb 19, 2010·Proceedings of the National Academy of Sciences of the United States of America·Nicola J BaxterJonathan P Waltho
Feb 28, 2002·Biochemistry·Patrick J O'Brien, Daniel Herschlag
Sep 30, 2006·Protein Science : a Publication of the Protein Society·Jie Wang, Evan R Kantrowitz
Jan 1, 2008·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Sandra K Grunwald, Katherine J Krueger
Feb 12, 2010·Dalton Transactions : an International Journal of Inorganic Chemistry·Jin-Tao WangLiang-Nian Ji
May 20, 2003·Nature Biotechnology·Sandro Cesaro-TadicAndreas Plückthun
Jan 30, 2015·Protein Science : a Publication of the Protein Society·Heather R BrodkinMary Jo Ondrechen
Apr 23, 2003·The Journal of Biological Chemistry·Robert R Boulanger, Evan R Kantrowitz
Jun 30, 2000·Journal of Molecular Biology·B StecE R Kantrowitz
Jun 27, 2002·Journal of Molecular Biology·Maaike de BackerEdward Hough
Jul 25, 2006·International Journal of Biological Macromolecules·S OrhanovićV Gamulin
Jan 3, 2007·Journal of Molecular Biology·Ellen WangPirkko Heikinheimo
Jan 8, 2021·Biochemistry·Ananda K Ghosh, Vern L Schramm
Nov 4, 2008·Biochimica Et Biophysica Acta·Ronny HellandBjarni Asgeirsson

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