Identification of a residue involved in transition-state stabilization in the ATPase reaction of DNA gyrase.
Examination of the X-ray crystal structure of the 43 kDa N-terminal domain of the DNA gyrase B protein (GyrB) shows that the majority of the interactions with bound ATP are made with subdomain 1 (residues 2-220). However, two residues from subdomain 2, Gln335 and Lys337, interact with the gamma-phosphate of ATP. The proposed roles for these residues include nucleotide binding, transition-state stabilization, and triggering protein conformational changes. We have used site-directed mutagenesis to convert Gln335 to Asn and Ala and Lys337 to Gln and Ala in the N-terminal domain of GyrB. Two of the resultant mutant proteins, GyrB43(Q335A) and GyrB43(K337Q), were shown to be correctly folded, and their interactions with ATP have been analyzed in detail. The Q335A protein is apparently unchanged with regard to nucleotide binding and hydrolysis, whereas the K337Q protein shows a modest decrease in nucleotide binding and a drastic reduction in ATPase activity. This is manifested by a approximately 10(3)-fold decrease in kcat. When the two mutations were moved into full-length GyrB, the Q335A mutation again showed little or no effect on activity, whereas the K337Q mutation had undetectable supercoiling and ATPase activities. We conclude that Gln335 is dispensable for ATP binding and hydrolysis by the gyrase B protein, whereas Lys337 has a critical role in the ATPase reaction and is likely to be a key residue in transition-state stabilization.
Smith
C V
CV
Department of Biochemistry, University of Leicester, UK.
Maxwell
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D002942:Circular Dichroism; D003374:Coumarins / Q000378:metabolism; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004120:Dimethyl Suberimidate; D004795:Enzyme Stability / Q000235:genetics; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics
D003374:Coumarins; D003432:Cross-Linking Reagents; D005973:Glutamine; D000266:Adenylyl Imidodiphosphate; D004120:Dimethyl Suberimidate; D000255:Adenosine Triphosphate; C030123:coumarin; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D008239:Lysine