Identification of a catalytic aspartyl residue of D-ribulose 5-phosphate 3-epimerase by site-directed mutagenesis.
Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.
Chen
Y R
YR
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831, USA.
Larimer
F W
FW
Serpersu
E H
EH
Hartman
F C
FC
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D017931:DNA Primers; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary
D017931:DNA Primers; D001224:Aspartic Acid; D002238:Carbohydrate Epimerases; C022553:ribulosephosphate 3-epimerase