Sugar Recognition by the Glucose and Mannose Permeases of Escherichia coli. Steady-State Kinetics and Inhibition Studies

Abstract

The glucose (EII(Glc)) and mannose (EII(Man)) permeases of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Escherichia coli belong to structurally different families of PTS transporters. The sugar recognition mechanism of the two transporters is compared using as inhibitors and pseudosubstrates all possible monodeoxy analogues, monodeoxyfluoro analogues, and epimers of D-glucose. The analogues were tested as phosphoryl acceptors in vitro and as uptake inhibitors with intact cells. Both EII have a high K(m) of phosphorylation for glucose modified at C-4 and C-6, and these analogues also are weak inhibitors of uptake. Conversely, modifications at C-1 (and also at C-2 with EII(Man)) were well tolerated. OH-3 is proposed to interact with hydrogen bond donors on EII(Glc) and EII(Man), since only substitution by fluorine was tolerated. Glucose-6-aldehydes, which exist as gem-diols in aqueous solution, are potent and highly selective inhibitors of "nonvectorial" phosphorylation by EII(Glc) (K(I) 3-250 microM). These aldehydes are comparatively weak inhibitors of transport by EII(Glc) and of phosphorylation and transport by EII(Man). Both transporters display biphasic kinetics (with glucose and some analogues) but simple Michaelis-Menten kinetics with 3-fluoroglucose (and other analogues). Kinetic simulations of the phosphorylation activities measured with different substrates and inhibitors indicate that two independent activities are present at the cytoplasmic side of the transporter. A working model that accounts for the kinetic data is presented.

Structure and function of the glucose PTS transporter from Escherichia coli

“…Saturation experiments of wt-IICB with increasing concentrations of [ 3 H]glucose revealed a K D of $50 lM (Fig. 3B) in agreement with previous results: 3-220 lM (Garcia-Alles et al, 2002;Postma et al, 1993). Substrate specificity of wt-IICB was explored by competitive inhibition of [ 3 H]glucose binding (Fig.…”

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

“…Garcia-Alles et al (18,33) reported that IICB Glc from E. coli exhibits steady-state kinetics that are biphasic when a large range of sugar concentrations (50 M to 5 mM) is tested. The authors attribute this to the presence of at least three (and perhaps four) catalytic sites that fall into two classes, one with higher affinity for Glc (K S ϳ 10 M) but lower phosphorylation activity and the other with low affinity for Glc (K S ϳ300 M) but about 6 times the phosphorylation activity of the high affinity class.…”

Direct, Mild, and Selective Synthesis of Unprotected Dialdo‐Glycosides

“…These structures have proved advantageous for a variety of reactions, including reductive amination protocols to produce N-linked glycosides and glycoconjugates, [6,7] glycoside oxime formation, [8] olefination reactions [9] and different polymerization protocols. [10,11] The synthesis of 1,5-dialdo-pyranosides has been addressed over the years, and some enzymatic, [12][13][14] as well as several synthetic, [12,15] pathways have been reported. The enzymatic procedures use galactose oxidase and are straightforward but are largely limited to galactose derivatives because of the enzymatic specificity for this structure.…”