THE ROLE OF AN ACTIVE-SITE LYSYL RESIDUE OF SPINACH PHOSPHORIBULOKINASE AS EXPLORED BY SITE-DIRECTED MUTAGENESIS

Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycl e enzyme phosphoribulokinase (PRK) from spinach has been assigned to t he active-site region [Miziorko et al. (1990), J. Biol. Chem. 265, 364 2-36471. The equivalent position is occupied by lysyl or arginyl resid ues in the PRK from both prokaryotic and eukaryotic sources, suggestin g a requirement for a basic residue at this location. To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl , glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesi s. All of the mutant enzymes retain substantial kinase activity; and e ven in the case of the radical substitution by glutamate, the K(m) val ues for ATP and ribulose 5-phosphate are not perturbed significantly. Glutamate at position-68 may destabilize tertiary structure, because t he yield of this mutant protein from transformed E. coli is quite low compared to that of the other proteins in this series. Despite the act ive-site proximity of Lys68, our results show that this residue does n ot play a key role in catalysis or substrate binding.