NMR-STUDIES OF THE CONFORMATIONS AND LOCATION OF NUCLEOTIDES BOUND TOTHE ESCHERICHIA-COLI MUTT ENZYME

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates t o nucleoside monophosphates and pyrophosphate by substitution at the r arely attacked beta-phosphorus. Nucleotides containing bulky substitue nts at the 8 position of the purine ring are preferentially hydrolyzed . The conformation of the MutT-bound nonhydrolyzable substrate analog Mg2+-AMPCPP, determined by 10 intramolecular NOEs and molecular dynami cs refinement using a full relaxation matrix analysis with back-calcul ation of the NOESY intensities, is high anti (chi = 53 +/- 9 degrees), with a C2'-exo, O1'-endo sugar pucker. Similarly, the product of dGTP hydrolysis, dGMP, also binds MutT in a high anti (chi = 73 +/- 9 degr ees) C1'-endo conformation based on seven intramolecular NOEs. Such hi gh anti rotations of the base would allow MutT to accommodate nucleoti des substituted at the C-8 position with no intramolecular clashes. Ch anges in chemical shifts in the H-1-N-15 spectra of the enzyme induced by Mg2+ and Mg(2+)AMPCPP suggest that the metal activator and nucleot ide interact with residues in loop I, at the carboxyl end of helix I, loop II, loop LII, and beta-strands A and B of the secondary structure of MutT. The displacement of Mg2+ by Mn2+ causes the selective disapp earance due to paramagnetic broadening of H-1-N-15 cross peaks from G3 7, G38, and K39 in loop I and E57 in helix I. Eleven intermolecular NO Es between Mg(2+)AMPCPP and hydrophobic residues of MutT are found, th ree of which are tentatively assigned to L67 in loop II and three to L 54 in helix I. Similarly, seven intermolecular NOEs between dGMP and h ydrophobic residues of the enzyme are found, four of which are tentati vely assigned to L54 and two to V58, both in helix I. These interactio ns indicate that the loop I-helix I-loop II motif contributes signific antly to the active site of MutT in accord with mutagenesis studies an d with sequence homologies among MutT-like NTP pyrophosphohydrolases.