NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.
The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.
Weber
D J
DJ
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Bhatnagar
S K
SK
Bullions
L C
LC
Bessman
M J
MJ
Mildvan
A S
AS
D001426:Bacterial Proteins / Q000378:metabolism*; D004260:DNA Repair; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010103:Oxygen Isotopes; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; D014867:Water
D001426:Bacterial Proteins; D003848:Deoxyguanine Nucleotides; D011756:Diphosphates; D029968:Escherichia coli Proteins; D010103:Oxygen Isotopes; D014867:Water; C007257:2'-deoxyguanosine 5'-phosphate; C029603:deoxyguanosine triphosphate; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C052211:mutT protein, E coli