Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP.
McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.
Pieper
U
U
Institut für Biochemie (FB 15), Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
Brinkmann
T
T
Krüger
T
T
Noyer-Weidner
M
M
Pingoud
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C069326:mcrC protein, E coli; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease; C059678:mcrB protein, E coli; D020558:GTP Phosphohydrolases