IIA^Glc Allosteric Control of Escherichia coli Glycerol Kinase:  Binding Site Cooperative Transitions and Cation-Promoted Association by Zinc(II)

Abstract

The catalytic activity of glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inhibited allosterically by IIA(Glc) (previously known as III(Glc)), the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system. A sequentially contiguous portion of glycerol kinase undergoes an induced fit conformational change involving coil, alpha-helix, and 3(10)-helix upon IIA(Glc) binding. A second induced fit occurs upon binding of Zn(II) to a novel intermolecular site, which increases complex stability by cation-promoted association. Eight of the ten sequentially contiguous amino acids are substituted with alanine to evaluate the roles of these positions in complex formation. Effects of the substitutions reveal both favorable and antagonistic contributions of the normal amino acids to complex formation, and Zn(II) reverses these contributions for two of the amino acids. The consequences of some of the substitutions for IIA(Glc) inhibition are consistent with changes in the intermolecular interactions seen in the crystal structures. However, for the amino acids that are located in the region that is alpha-helical in the absence of IIA(Glc), the effects of the substitutions are not consistent with changes in intermolecular interactions but with increased stability of the alpha-helical region due to the higher alpha-helix propensity of alanine. The reduced affinity for IIA(Glc) binding seen for these variants is consistent with predictions of Freire and co-workers [Luque, I., and Freire, E. (2000) Proteins: Struct., Funct., Genet. 4, 63-71]. These variants show also increased cation-promoted association by Zn(II) so that the energetic contribution of Zn(II) to complex formation is doubled. The similarity of effects of the alanine substitutions of the amino acids in the alpha-helical region for IIA(Glc) binding affinity and cation-promoted association by Zn(II) indicates that they function as a cooperative unit.

Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: Inhibition of E. coli glycerol kinase

“…The parameters K 0.5 and W that are obtained from the fits are shown in Table 1. The values for K 0.5 and W for EGK agree with those from earlier work [41]. The A65T and R369A amino acid substitutions change the apparent affinity for IIA Glc binding in the saturating presence of substrates as shown by the values for K 0.5 .…”

IIAGlc Inhibition of Glycerol Kinase: A Communications Network Tunes Protein Motions at the Allosteric Site

“…The maximum extent of IIA Glc inhibition (I max ) is reduced for the I474C and R479C variants. Similar results were obtained for the I474A and R479A variants (31). The small effects of these scanning cysteine substitutions on the catalytic and allosteric properties indicate that these variants should provide meaningful insights into the dynamics properties of the IIA Glc -binding site.…”

Experimental and Computational Assessment of Conditionally Essential Genes inEscherichia coli

“…PTS enzyme I, encoded by ptsI, is phosphorylated in a reaction with PEP in the first step of the PTS, and crr encodes PTS glucose-specific enzyme IIA (EIIA Glc ), which is another intermediate that transfers the PTS phosphate to glucose. EIIA Glc is also a central regulatory molecule in E. coli metabolism (35), and in its unphosphorylated form, EIIA Glc binds and allosterically inhibits GlpK, thus ultimately impeding glycerol uptake and metabolism (21,22). Phosphorylation of EIIA Glc releases GlpK, however, and facilitates normal glycerol uptake and metabolism.…”