THE CHLAMYDOMONAS-REINHARDTII CF1 WITH THE MUTATION BETA-T168S HAS HIGH ATPASE ACTIVITY

We have generated the mutation T168S in the beta subunit of the chloro plast ATP synthase complex of Chlamydomonas reinhardtii by site direct ed mutagenesis and chloroplast transformation, CF1 and the alpha(3) be ta(3) gamma complex: of this mutant strain were isolated and their enz ymatic activities were characterized and compared to those of the corr esponding wild type complexes, Without activation the mutant CF1 exhib its MgATPase activity with at least 10 times higher rates than the wil d type enzyme, The MgATPase activity could be stimulated to some exten t by methanol, but less by ethanol and octylglucoside, The alpha(3) be ta(3) gamma complex had an even higher MgATPase activity, which was on ly slightly enhanced by ethanol or methanol, The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg2+., Free ADP inhibited neither t he mutant nor the wild type ATPase significantly, Azide, which strongl y inhibited the ATPase activity of the mild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesti ng that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast A,MgADP activity, The m utant cells grew photoautotrophically at a growth rate of about 50%., Similar to the mild type the cells survived on minimal medium in the d ark, Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically, (C) 1998 Fed eration of European Biochemical Societies.