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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*

Shaun R. Brinsmade,J. Escalante‐Semerena

Published 2007 in Journal of Biological Chemistry

ABSTRACT

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 (kcat 2.5–3-fold > kcat PtaWT. Results from sedimentation equilibrium experiments are consistent with PtaWT being a trimer. Pta variants formed more hexamer than the PtaWT protein. NADH inhibited PtaWT activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein PtaR252H. Pyruvate stimulated PtaWT activity, and its effect was potentiated in the variants, being most pronounced on PtaR252H.

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