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

Yuh-Ru Chen,Frank W Larimer,Engin H Serpersu,Fred C Hartman

doi:10.1074/jbc.274.4.2132

Yuh-Ru Chen, Frank W Larimer + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.4.2132

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Abstract

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.

Highlights

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
Purity and Structural Integrity of Ru5P Epimerase Mutants—Electrophoretic analyses indicate a high degree of purity of the epimerase mutants, which comigrate with the wildtype enzyme during denaturing (Fig. 3A) and nondenaturing (Fig. 3B) PAGE
Because of the numerous enzymes that abstract protons ␣ to carbonyl or carboxyl groups, including D-ribose 5-phosphate isomerase that shares a common substrate with Ru5P epimerase and several others that transform substrates structurally similar to Ru5P, we expected to uncover some enzymes with regions of sequence similarity with Ru5P epimerase through data base searches

Summary

The abbreviations used are

Ru5P, ribulose 5-phosphate; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; Xu5P, xylulose 5-phosphate. The epimerase-catalyzed reaction probably entails a twobase mechanism with an enediolate intermediate (Fig. 1). This supposition is based on the observations that Ru5P formed from [3-2H]Xu5P was completely free of deuterium, even though deuterium was completely retained in the remaining Xu5P [2]. Because three-dimensional structural information and chemical modification data of Ru5P epimerase are unavailable to guide selections of site-directed mutants, we have assessed comparative sequences and predictive secondary structural analyses for candidate catalytic residues. Properties of the D186E, D186N, and D186A mutants of Ru5P epimerase are entirely compatible with a crucial catalytic role for Asp186

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION