DIFFERENTIAL-EFFECTS OF N-TERMINAL AND C-TERMINAL DELETIONS ON THE 2 ACTIVITIES OF RUBISCO ACTIVASE

Spinach (Spinacea oleracea) leaf ribulose-1,5-bisphosphate carboxylase /oxygenase (rubisco) activase was subjected to limited proteolysis wit h trypsin and directed deletions were made by modifying the spinach ru bisco activase cDNA and expressing the 41-kDa isoform in Escherichia c oli. Protein exposed to trypsin displayed a more rapid loss of the abi lity to promote the activation of decarbamylated rubisco than ATP hydr olysis (e.g., 10 and 50% activity remaining, respectively, after 1 h). A series of N-terminal deletions exhibited near abolition of rubisco activation after the 12th residue, a conserved tryptophan, was deleted . Conversely, a deletion of 19 residues at the C-terminus increased ru bisco activation with little effect on ATP hydrolysis, resulting in an increased efficiency of activation. The C-terminal deletion mutant wa s further modified by a site-directed mutation in the ATP binding regi on (Q109E) which was previously observed to increase the efficiency of activation (J. B. Shen and W. L. Ogren, 1991, Plant Physiol. 99, 1201 -1207). The efficiency of activation with this double mutant was great er than that for either of the original mutants. The results indicate that a conserved tryptophan in the N-terminal portion of rubisco activ ase is critical for promotion of the activation of rubisco, consistent with a possible role in interaction with rubisco. The C-terminus appe ars to have a regulatory effect on both rubisco activation and ATP hyd rolysis. (C) 1996 Academic Press, Inc.