ATPASE KINETICS FOR WILD-TYPE SACCHAROMYCES-CEREVISIAE F1-ATPASE AND F1-ATPASE WITH THE BETA-SUBUNIT THR197-]SER MUTATION

Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F-1-ATPase and F-1-ATPase with the Thrl97 --> Ser mutation in the beta subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high. The Thrl97 --> Ser mutation in the yeast F-1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F-1-ATPase for ADP is altered by azide, oxyanions and the Thrl97 --> Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thrl97 --> Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thrl97 --> Ser mutation as measured by determining the K-i(ADP) values for multisite ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under multisite conditions. Sulfite increases the K-i(ADP) values for ATP hydrolysis under multisite conditions approximately threefold for the wildtype and mutant enzymes and the Thrl97 --> Ser mutation increases K-i(ADP) ninefold. The effect of sulfite on K-i(ADP) is additive to the effect of the Thrl97 --> Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thrl97 --> Ser mutation on the biochemistry of F-1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thrl97 --> Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the K-i values. Furthermore, the mechanisms of activation by sulfite and the Thrl97 --> Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP.