SITE-DIRECTED MUTAGENESIS OF A REACTIVE LYSYL RESIDUE (LYS-247) OF RUBISCO ACTIVASE

Chemical modification of tobacco leaf ribulose-1,5-bisphosphate carbox ylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccini mide esters identified Lys-247 as a particularly reactive residue nece ssary for maximal catalytic activity [M. E. Salvucci (1993) Plant Phys iol. 103, 501-508]. To further explore the role of Lys-247 in catalysi s, this species-invariant residue of Rubisco activase was changed to A rg, Cys, and Gin by mutagenesis of a cDNA clone of the mature form of the tobacco enzyme. Analysis of the purified recombinant proteins show ed that all three point mutations reduced the rate of ATP hydrolysis t o 2 to 3% of the wild-type enzyme and completely abolished the ability of Rubisco activase to promote activation of decarbamylated Rubisco. Replacement of Lys-247 with Arg, Cys, or Gin had a comparatively minor effect on ATP binding, but eliminated the increase in ATPase-specific activity that normally occurs with increasing concentrations of Rubis co activase protein. in mixing experiments, the K247R mutant enzyme in hibited Rubisco activation by wild-type Rubisco activase, indicating t hat interactions between Rubisco and Rubisco activase were disrupted b y even the most conservative of the substitutions. Chemical elaboratio n of the K247C mutant by treatment with a-bromoethylamine converted 39 % of the thiols at position 247 to the aminoethyl derivative, but fail ed to improve the catalytic performance of the mutant enzyme. Our resu lts indicate that the requirement for a lysyl residue at position 247 of Rubisco activase is very stringent, consistent with its proposed ro le in coordinating precise interactions with gamma-phosphate of ATP. ( C) 1994 Academic Press, Inc.