CATALYTIC PROPERTIES AND SENSITIVITY TO TENTOXIN OF CHLAMYDOMONAS-REINHARDTII ATP SYNTHASES CHANGED IN CODON-83 OF ATPB BY SITE-DIRECTED MUTAGENESIS

The participation of the amino acid beta 83 in determining the sensiti vity of chloroplast ATP synthases to tentoxin was reported previously, We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene b y site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechan ism of ATP synthesis and hydrolysis, Amino acid beta 83 was changed fr om Glu to Asp (beta E83D) and to Lys (beta E83K), and the highly conse rved tetrapeptide beta T82-E83-G84-L85 (Delta TEGL) was deleted, Mutan t strains were produced by particle gun transformation of atpB deletio n mutants cw15 Delta atpB and FUD50 with the mutated atpB genes, The t ransformants containing the beta E83D and beta E83K mutant genes grew well photoautotrophically. The Delta TEGL transformant did not grow ph otoautotrophically, and no CF1 subunits were detected by immunostainin g of Western blots using CF1 specific antibodies, The rates of ATP syn thesis at clamped Delta pH with thylakoids isolated from cw15 and the two mutants, beta E83D and beta E83K, were similar, However, only the phosphorylation activity of the mutant beta E83D was inhibited by tent oxin with 50% inhibition attained at 4 mu M. These results confirm tha t amino acid beta 83 is critical in determining the response of ATP sy nthase to tentoxin, The rates of the latent Mg-ATPase activity of the CF(1)s isolated from cw15, beta E83D, and beta E83K were similar and c ould be enhanced by heat, alcohols, and octylglucoside, As in the case of the membrane-bound enzyme, only CF1 from the beta E83D mutant was sensitive to tentoxin, A lower alcohol concentration was required for optimal stimulation of the ATPase of the beta E83K-CF1 than that of CF 1 from the other two strains, Moreover, the optimal activity of the be ta E83K-CF1 was also lower, These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the ''crown'' domain affects the active conformation of the CF1-ATPase .