Mechanism of phosphatase activity in the chemotaxis response regulator CheY.
Response regulator proteins are phosphorylated on a conserved aspartate to activate responses to environmental signals. An intrinsic autophosphatase activity limits the duration of the phosphorylated state. We have previously hypothesized that dephosphorylation might proceed through an intramolecular attack, leading to succinimide formation, and such an intramolecular dephosphorylation event is seen for CheY and OmpR during mass spectrometric analysis [Napper, S., Wolanin, P. M., Webre, D. J., Kindrachuk, J., Waygood, B., and Stock, J. B. (2003) FEBS Lett 538, 77-80]. Succinimide formation is usually associated with the spontaneous deamidation of Asn residues. We show here that an Asp57 to Asn mutant of the CheY chemotaxis response regulator undergoes an unusually rapid deamidation back to the wild-type Asp57, supporting the hypothesis that the active site of CheY is poised for succinimide formation. In contrast, we also show that the major route of phosphoaspartate hydrolysis in CheY occurs through water attack on the phosphorus both during autophosphatase activity and during CheZ-mediated dephosphorylation. Thus, CheY dephosphorylation does not usually proceed via a succinimide or any other intramolecular attack.
Wolanin
Peter M
PM
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Webre
Daniel J
DJ
Stock
Jeffry B
JB
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002633:Chemotaxis / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism
D000577:Amides; D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010103:Oxygen Isotopes; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D013388:Succinimides; D001224:Aspartic Acid; D001216:Asparagine; D010744:Phosphoric Monoester Hydrolases