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J. Biol. Chem., Vol. 282, Issue 17, 12629-12640, April 27, 2007

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In Vivo and in Vitro Analyses of Single-amino Acid Variants of the Salmonella enterica Phosphotransacetylase Enzyme Provide Insights into the Function of Its N-terminal Domain^*

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

The function of the N-terminal domain (350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mM acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (k_cat 2.5–3-fold > k_cat Pta^WT. Results from sedimentation equilibrium experiments are consistent with Pta^WT being a trimer. Pta variants formed more hexamer than the Pta^WT protein. NADH inhibited Pta^WT activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein Pta^R252H. Pyruvate stimulated Pta^WT activity, and its effect was potentiated in the variants, being most pronounced on Pta^R252H.

Received for publication, December 13, 2006 , and in revised form, February 27, 2007.

^* This work was supported in part by Public Health Service Grant R01-GM62203 (to J. C. E.-S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 4 and 5 and Figs. 8–11.

¹ Supported in part by the Ira Baldwin Wisconsin Distinguished Predoctoral Fellowship (from the Department of Bacteriology) and the Thomsen Wisconsin Distinguished Predoctoral Fellowship (from the College of Agricultural and Life Sciences).

² To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 1550 Linden Dr., Madison, WI 53706. Tel.: 608-262-7379; Fax: 608-265-7909; E-mail: escalante{at}bact.wisc.edu.

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