NMR studies of the conformations and location of nucleotides bound to the Escherichia coli MutT enzyme.

摘要: The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates to monophosphates and pyrophosphate by substitution at rarely attacked beta-phosphorus. Nucleotides containing bulky substituents 8 position purine ring are preferentially hydrolyzed. conformation MutT-bound nonhydrolyzable substrate analog Mg(2+)-AMPCPP, determined 10 intramolecular NOEs molecular dynamics refinement using a full relaxation matrix analysis with back-calculation NOESY intensities, is high anti (chi = 53 +/- 9 degrees), C2'-exo, O1'-endo sugar pucker. Similarly, product dGTP hydrolysis, dGMP, also binds in 73 degrees) C1'-endo based on seven NOEs. Such rotations base would allow accommodate nucleotides substituted C-8 no clashes. Changes chemical shifts 1H-15N spectra induced Mg2+ AMPCPP suggest that metal activator nucleotide interact residues loop I, carboxyl end helix II, III, beta-strands A B secondary structure MutT. displacement Mn2+ causes selective disappearance due paramagnetic broadening cross peaks from G37, G38, K39 I E57 I. Eleven intermolecular between Mg2+AMPCPP hydrophobic found, three which tentatively assigned L67 II L54 dGMP four two V58, both These interactions indicate I-helix I-loop motif contributes significantly active site accord mutagenesis studies sequence homologies among MutT-like NTP pyrophosphohydrolases.