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

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

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

J Biol Chem. 1992 Aug 25;267(24):16939-42

Authors: Weber DJ, Bhatnagar SK, Bullions LC, Bessman MJ, Mildvan AS

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.

PMID: 1324915 [PubMed - indexed for MEDLINE]



Source: PubMed