EVIDENCE SUPPORTING CATALYTIC ROLES FOR ASPARTATE RESIDUES IN PHOSPHORIBULOKINASE

The DNA encoding Rhodobacter sphaeroides phosphoribulokinase (PRK) has been modified to allow ligation into pET-3d. Using the resulting expr ession plasmid, PRK was overexpressed in Escherichia coli and isolated in milligram quantities. Homogeneous preparations of the enzyme exhib it properties comparable to those of PRK expressed using a previously described pUC19-derived construct [Sandbaken et al., Biochemistry 31, 3715-3719]. Mutagenesis experiments have been designed to produce cons ervative substitutions that eliminate the carboxyl groups of each of f our conserved acidic residues (D42, E131, D169, and E178). Using the n ewly developed expression system, the resulting PRK variants have been expressed, isolated, and characterized. Expression levels and recover ies upon affinity chromatography purification are similar to the resul ts obtained with wild-type PRK. Apparent substrate affinities of these mutant proteins do not differ greatly from values observed for wild-t ype PRK. In contrast, these PRK variants display a wide range of V-max values, ranging from wild-type activity (similar to 200 units/mg; E17 8A) to levels that are diminished by 4 (D169A) to 5 (D42A, D42N) order s of magnitude. That the large diminutions in catalytic activity are s ignificant and do not merely reflect gross perturbations in protein st ructure is suggested not only by the modest effects on substrate affin ity but also by the allosteric properties of D169A, D42A, and D42N. Th e activities of these proteins, like that of wild-type PRK, are marked ly stimulated by the positive effector NADH. The magnitude of the V-ma x perturbations suggests that D42 and D169 are candidates for the role of active site base or activator cation ligand. In contrast to the ma rked diminution of V-max observed upon mutation of D42 or D169, only a 2 order of magnitude decrease is observed with E131A; much of this ef fect may be attributed to the fact that this variant no longer is sens itive to allosteric stimulation by NADH.