Mutagenesis and reversion analysis of residue Met-209 of the beta-subunit of Escherichia coli ATP synthase.
Residue beta-Met-209 is conserved in all known F1-ATPase sequences, and the mutation beta M209I in Escherichia coli causes profound inhibition of ATP synthesis and hydrolysis. Based on the properties of this mutant it had previously been proposed that residue beta-209 lies close to the site of catalysis. Two approaches were used to study this residue further. First, revertants were sought. Only wild-type and beta-Ser-209 were found; the Ser revertants involved a two-base change. Significantly, Ser is found at the equivalent position in the homologous vacuolar and archaebacterial ATPases. Second, all 20 natural amino acids were placed at position beta-209 by mutagenesis, and catalytic properties of the mutants were analyzed. The results showed that only a limited set of residues supported significant growth or ATPase activity, and that many of the mutations impacted severely on catalysis. X-ray structure analysis of the bovine enzyme has revealed that residue beta-Met-209 lies only 3.1. A from residue beta-Glu-181, which has been proposed to act as catalytic base. The results reported here emphasize that, in this discrete region of the catalytic site, specific stereo-chemical constraints on structure are critical for catalysis.
Wilke-Mounts
S
S
Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
Pagan
J
J
Senior
A E
AE
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D015151:Immunoblotting; D008566:Membranes / Q000201:enzymology; D008715:Methionine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D017494:Proton Pumps; D000255:Adenosine Triphosphate; D008715:Methionine; D006180:Proton-Translocating ATPases