A cDNA encoding @entity2772 chloroplastic @entity17457 isomerase (RPI) was cloned and overexpressed in @entity526 , and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from @entity2772 leaves (A. C. Rutner, 1970, Biochemistry 9, 178-184). @entity1391 , used as a buffer in previous studies, is a competitive inhibitor of RPI with a K(i) of 7.9 mM. @entity69472 is an effective inhibitor, while @entity17456 is not, indicating that the configuration at @entity505 -3 contributes to substrate recognition. Although @entity69472 binds to RPI, it is not isomerized, demonstrating that the configuration at @entity505 -2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged @entity535 residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. @entity69473 , @entity69474 , and @entity69475 mutants show greatly diminished k(cat) values (0. 0012, 0.074, and 0.38% of the wild type, respectively), while @entity69476 retains substantial activity. Only insignificant or moderate changes in K(m) of @entity17457 are observed for @entity69473 , @entity69474 , and @entity69475 , indicating a direct or indirect catalytic role of the targeted residues.

XXXX isomerase from @entity2772 : heterologous overexpression, purification, characterization, and site-directed mutagenesis of the recombinant enzyme.

@entity17457 :: ('MESH:C031626', 'Chemical') :: ['ribose-5-phosphate', 'D-ribose 5-phosphate'] @entity69474 :: ('p|SUB|K|100|A', 'ProteinMutation') :: ['K100A'] @entity69473 :: ('p|SUB|D|87|A', 'ProteinMutation') :: ['D87A'] @entity2772 :: ('3562', 'Species') :: ['spinach'] @entity535 :: ('CHEBI:33704', 'Chemical') :: ['amino acid'] @entity69476 :: ('p|SUB|E|91|A', 'ProteinMutation') :: ['E91A'] @entity505 :: ('MESH:D002244', 'Chemical') :: ['carbon'] @entity1391 :: ('CHEBI:26020', 'Chemical') :: ['Phosphate'] @entity69475 :: ('p|SUB|D|90|A', 'ProteinMutation') :: ['D90A'] @entity69472 :: ('MESH:C028055', 'Chemical') :: ['D-arabinose 5-phosphate', 'D-Arabinose 5-phosphate'] @entity526 :: ('562', 'Species') :: ['Escherichia coli'] @entity17456 :: ('MESH:C031625', 'Chemical') :: ['D-xylulose-5 phosphate']
@entity17457 :: (MESH:C031626,Chemical) :: ['D-ribose-5-phosphate']
