Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation.

Abstract

In enteric bacteria, the phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the uptake and phosphorylation of its sugar substrates. In addition, the PTS regulates adenylate cyclase, glycerol kinase (GK), and a number of non-PTS permeases (for reviews, see refs. 1-4). In the present report, we show that the binding of IIIic to GK generates a putative intermolecular zinc coordination site. Zn(II) not only specifically binds at this site but substantially increases the apparent affinity of the two proteins and the inhibitory potency of 1IIGIc for GK. These results have significant implications for the mechanisms of regulation of multiple proteins by IIIiGC. MATERIALS AND METHODSCrystals ofthe IIIGIc/GK complex were prepared by hangingdrop vapor diffusion as described (8) and transferred to storage buffer containing 20 mM cation chloride, 20 mM ADP, 50 mM glycerol 3-phosphate (G3P), and 0.6 M sodium acetate in 100 mM Mes buffer, pH 6.1. Normally, all solutions were prepared in ultrafiltered water (Nanopure II; Barnstead), but the MgCl2 solution described below was inadvertently prepared with reverse-osmosis water (15-fold higher conductivity than the ultrafiltered water).Diffraction data using graphite-monochromated CuKa radiation were collected on a San Diego Multiwire Systems area detector and reduced by the supplied software. Crystallographic refinement was performed with the TNT package of programs (9), and electron density maps were inspected with FRODO (10).A site-directed mutant of 111GIc, H75Q (His75 -* Gln), was prepared as reported (11). GK assays were conducted (12) with the homogeneous enzyme in the presence or absence of IJIGic and Zn(II) as indicated. The effect of Zn(II) on the coupling enzyme system was investigated. ZnADP is a substrate for pyruvate kinase, but the efficacy of the coupling system is not affected by the Zn(II) concentrations used in these experiments. The specific activity of GK is constant over the range of enzyme concentrations (0.5-2.5 pg/ml) used with 0.1 mM ZnCl2 added to the assay mixture.Abbreviations: GK, glycerol kinase; PTS, phosphotransferase system; G3P, glycerol 3-phosphate.$To whom reprint requests should be sent at the * address. 3544The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo

“…The parameters obtained from the fit show that binding of III Glc gives 95% inhibition of the wild-type enzyme but gives twofold, i.e., 100%, activation of the G-304-S enzyme. For the wild-type enzyme, the apparent dissociation constants for III Glc binding are 15 M in the absence of Zn(II) and 0.6 M with 0.1 mM Zn(II), values which agree with our previous report (5). For the G-304-S enzyme, the corresponding values are 110 and 9 M. Thus, the G-304-S substitution decreases the apparent affinity for III In the related protein DnaK, conformational change due to ATP binding affects cleavage by proteases (9).…”

“…We are investigating the molecular basis of these allosteric control mechanisms by which glycerol kinase functions in a signal transduction pathway that modulates gene expression in response to carbon source availability. The crystal structure of the complex of glycerol kinase with the unphosphorylated form of III Glc has been determined (8), and the association of the two proteins forms a novel intermolecular binding site for Zn(II) (5). A proposed mechanism for FBP regulation postulates that FBP binds to and inhibits the tetrameric form of the enzyme (3).…”

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Indeed, crystals soaked in a 20 mM ZnCl 2 solution adopted a Zn 2ϩ ion at the expected position (223). Subsequent in vitro studies revealed that the presence of the metal ion lowers the K i of EIIA Glc for GlpK by about 60-fold (223,616 It is not known how the interaction with unphosphorylated EIIA Glc inhibits glycerol phosphorylation. The more than 30-Å distance between the sites of EIIA Glc binding and glycerol phosphorylation rules out a direct effect.…”

Understanding Glucose Transport by the Bacterial Phosphoenolpyruvate:Glycose Phosphotransferase System on the Basis of Kinetic Measurements in Vitro

“…The question arises how the PTS can still regulate other systems by inducer exclusion under these conditions, because unphosphorylated IIA Glc binds stoichiometrically to its target proteins. Additional simulations (54) have shown that introduction of a step for binding of free unphosphorylated IIA Glc to a target protein leads to a redistribution of the IIA Glc forms and that a significant fraction of the total IIA Glc can bind to the target protein if the dissociation equilibrium constant is in the range of 0.2-1 M, as reported for glycerol kinase and IIA Glc in the presence of Zn(II) (55). However, the addition of glucose by itself to a suspension of E. coli cells (i.e.…”