Site-directed Mutagenesis of the Phosphocarrier Protein. IIIGlc, a Major Signal-transducing Protein in Escherichia Coli

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1989 Jun 1

PMID 2657735

Citations 17

Authors

K A Presper

C Y Wong

L Liu

N D Meadow

S Roseman

Affiliations

Soon will be listed here.

Abstract

The glucose-specific phosphocarrier protein (IIIGlc) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is a major signal transducer that mediates the intricate interplay among extracellular signals (PTS and non-PTS sugars), cytoplasmic and membrane proteins (PTS and non-PTS transporters), and adenylate cyclase. To further define the central role of IIIGlc in these multiplex signaling mechanisms, we have used site-directed mutagenesis to construct three mutant IIIGlc proteins containing single amino acid changes; Phe-3 was replaced with tryptophan [( Trp3]IIIGlc), and His-75 and the active-site His-90 were replaced with glutamine [( Gln75]IIIGlc and [Gln90]IIIGlc, respectively). [Trp3]IIIGlc resembles the wild-type protein in most properties and should be valuable for spectrophotometric experiments. In contrast, clear differences between mutant and wild-type proteins were observed with both [Gln75]IIIGlc and [Gln90]IIIGlc in in vitro sugar phosphorylation assays. As predicted, [Gln90]IIIGlc with a modified active site cannot be phosphorylated. Unexpectedly, [Gln75]IIIGlc accepts but cannot transfer phosphoryl groups, suggesting His-75 may also be a critical amino acid for IIIGlc-mediated signaling mechanisms. The physiological effects of these mutations are briefly described.

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