Information Transfer in Multienzyme Complexes

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Abstract
A mutant phosphoribulokinase has been isolated from the 12‐2B mutant of Chlamydomonas reinhardtii. In this mutant, Arg64 has been replaced by Cys. The enzyme, which may exist in the dimeric and tetrameric states, is almost devoid of activity. Neither of these enzymes is able to form a complex with glyceraldehyde‐3‐phosphate dehydrogenase. The phosphoribulokinase gene has been expressed in Escherichia coli. The resulting recombinant protein, after isolation and purification, is apparently identical to the native enzyme extracted from the chloroplast. Three mutants have been generated by site directed mutagenesis. Arg64 has been replaced by Ala, Lys or Glu. With the exception of the latter, the two other mutants, [A64]phosphoribulokinase and [K64]phosphoribulokinase, 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 wild‐type recombinant phosphoribulokinase. Both the catalytic constant and the apparent affinity of ribulose 5‐phosphate are decreased relative to the corresponding values obtained for the wild‐type, the native or the recombinant enzyme. Whereas the [A64]phosphoribulokinase is unable to form a complex with glyceraldehyde‐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]phosphoribulokinase becomes active in the presence of glyceraldehyde‐3‐phosphate dehydrogenase but this activity is smaller than that of the corresponding wild‐type enzyme. Taken together, these results show that Arg64 plays a major role in the association of the two enzymes and in the information 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 that it stabilizes a conformation that is required for both the catalytic activity and the formation of the bienzyme complex.