NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction

• Published in: The Journal of biological chemistry 1992-08, Vol.267 (24), p.16939-16942
• Main Authors: Weber, D J, Bhatnagar, S K, Bullions, L C, Bessman, M J, Mildvan, A S
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - metabolism; Deoxyguanine Nucleotides - metabolism; Diphosphates - metabolism; DNA Repair; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins; Hydrolysis; Isotope Labeling - methods; Magnetic Resonance Spectroscopy - methods; Oxygen Isotopes; Phosphoric Monoester Hydrolases; Pyrophosphatases; Water
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)41875-3

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.