INFORMATION-TRANSFER IN MULTIENZYME COMPLEXES .2. THE ROLE OF ARG64 OF CHLAMYDOMONAS-REINHARDTII PHOSPHORIBULOKINASE IN THE INFORMATION-TRANSFER BETWEEN GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND PHOSPHORIBULOKINASE

A mutant phosphoribulokinase has been isolated from the 12-2B mutant o f Chlamydomonas reinhardtii. In this mutant, Arg64 has been replaced b y Cys. The enzyme, which may exist in the dimeric and tetrameric state s, is almost devoid of activity. Neither of these enzymes is able to f orm a complex with glyceraldehyde-3-phosphate dehydrogenase. The phosp horibulokinase gene has been expressed in Escherichia coli. The result ing recombinant protein, after isolation and purification, is apparent ly identical to the native enzyme extracted from the chloroplast. Thre e mutants have been generated by site directed mutagenesis. Arg64 has been replaced by Ala, Lys or Glu. With the exception of the latter, th e two other mutants, [A64]phosphoribulokinase and [K64]phosphoribuloki nase, are active when they are reduced, and nearly totally inactive in their oxidized state. Their activity, however, is decreased relative to that of the native, or to that of the wildtype recombinant phosphor ibulokinase. Both the catalytic constant and the apparent affinity of ribulose 5-phosphate are decreased relative to the corresponding value s obtained for the wild-type, the native or the recombinant enzyme. Wh ereas the [A64]phosphoribulokinase is unable to form a complex with gl yceraldehyde-3-phosphate dehydrogenase, [K64]phosphoribulokinase does, but the stability of the resulting complex is much decreased relative to that of the wild-type complex. The oxidized mutant [K64]phosphorib ulokinase becomes active in the presence of glyceraldehyde-3-phosphate dehydrogenase but this activity is smaller than that of the correspon ding wild-type enzyme. Taken together, these results show that Arg64 p lays a major role in the association of the two enzymes and in the inf ormation transfer that takes place between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. As this residue also appears to be important for catalytic activity, it may be tempting to consider t hat it stabilizes a conformation that is required for both the catalyt ic activity and the formation of the bienzyme complex.