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 F-1-ATPase sequences, a nd the mutation beta M209I ire Escherichia coli causes profound inhibi tion of ATP synthesis and hydrolysis. Based on the properties of this mutant it had previously been proposed that residue beta-209 lies clos e to the site of catalysis. Two approaches were used to study this res idue further, First, revertants were sought. Only wild-type and beta-S er-209 were found; the Ser revertants involved a two-base change. Sign ificantly, Ser is found at the equivalent position in the homologous v acuolar and archaebacterial ATPases. Second, all 20 natural amino acid s were placed at position beta-209 by mutagenesis, and catalytic prope rties of the mutants were analyzed. The results showed that only a lim ited set of residues supported significant growth or ATPase activity, and that many of the mutations impacted severely on catalysis. X-ray s tructure analysis of the bovine enzyme has revealed that residue beta- Met-209 lies only 3.1 Angstrom from residue beta-Glu-181, which has be en proposed to act as catalytic base. The results reported here emphas ize that, in this discrete region of the catalytic site, specific ster eochemical constraints on structure are critical for catalysis (C) 199 5 Academic Press, Inc.