alpha-Aspartate 261 is a key residue in noncatalytic sites of Escherichia coli F1-ATPase.
X-ray structure analysis of the noncatalytic sites of F1-ATPase revealed that residue alpha-Asp261 lies close to the Mg of bound Mg-5'-adenylyl-beta,gamma-imidodiphosphate. Here, the mutation alpha D261N was generated in Escherichia coli and combined with the alpha R365W mutation, allowing nucleotide binding at F1 noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified alpha D261N/alpha R365W F1-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleotide-depletion procedures, the noncatalytic sites in purified native enzyme were already empty. Binding studies with MgATP, MgADP, and the corresponding free nucleotides led to the following conclusions. Residue alpha-Asp261 interacts with the Mg of Mg-nucleotide in noncatalytic sites and provides a large component of the binding energy (approximately 3 kcal/mol). It is the primary determinant of the preference of noncatalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind poorly. Under conditions where noncatalytic sites were empty, alpha D261N/alpha R365W F1 showed significant hydrolysis of MgATP. This established unequivocally that occupancy of noncatalytic sites by nucleotide is not required for catalysis.
Weber
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
Bowman
C
C
Wilke-Mounts
S
S
Senior
A E
AE
D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases