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J Biol Chem, Vol. 274, Issue 4, 2132-2136, January 22, 1999

Identification of a Catalytic Aspartyl Residue of D-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Yuh-Ru Chen, Frank W. Larimer^§, Engin H. Serpersu, and Fred C. Hartman^§

From the ^§ Protein Engineering Program, Life Sciences Division, Oak Ridge National Laboratory, and  University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831 and  Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996

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 k_cat of 7.1 × 10³ s¹ is lowered to 3.3 × 10⁴ s¹ in D186A, 0.13 s¹ in D186N, and 1.1 s¹ in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the -carboxyl of Asp¹⁸⁶ facilitates catalysis by >10⁷-fold. Relative to the wild-type enzyme, the K_m 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 -proton abstraction. This study is the first to identify a catalytic group of the epimerase.

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