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Journal of Bacteriology, August 2003, p. 4837-4843, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4837-4843.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis

T. L. Grimek,¹ H. Holden,² I. Rayment,² and J. C. Escalante-Semerena^1*

Departments of Bacteriology,¹ Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin²

Received 10 March 2003/ Accepted 13 May 2003

The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg²⁺ ions; equimolar concentrations of Mn²⁺ ions were a poor substitute for Mg²⁺ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K_m of PrpB for 2-MIC was measured at 19 µM, with a k_cat of 105 s^-1. Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB^K121A, PrpB^H125A, and PrpB^R122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k_cat for 2-MIC lyase activity, respectively. The PrpB^D58A and PrpB^C123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg²⁺ ion.

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* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin, 1710 University Ave., Madison, WI 53726-4087. Phone: (608) 262-7379. Fax: (608) 265-7909. E-mail: escalante{at}bact.wisc.edu.

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Journal of Bacteriology, August 2003, p. 4837-4843, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4837-4843.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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