The Biology of Cyclic Nucleotides in E coli

Peterkofsky, A.

Abstract

A. Site-specific Mutagenesis of Presumptive ATP Binding Sites in Escherichia coli Adenylyl Cyclase. In addition to the region of the enzyme previously studied, two potential regions of interest were subjected to site-directed mutagenesis treatments. The focus of attention was on possibly essential lysine residues in these regions. It was found that the change of lysine 90 to either methionine or glutamine had a minor effect on the Vmax and that the Km for the glutamine mutant was reduced by a factor of 3. In contrast, the change of lysine 196 to methionine eliminated the activity and the change to glutamine reduced the activity by one order of magnitude and the Km by a factor of 3. B. Stimulation of Adenylyl Cyclase Activity by Nucleotides. Permeable cells, but not extracts, of E. coli contain adenylyl cyclase in a form that is stimulated by GTP as well as UTP and CTP. The kinetic properties of the enzyme are consistent with the interpretation that the nucleotides act as allosteric regulators of the activity. Mixing studies support the conclusion that there is a single site for all the active nucleotides. The expression of the allosteric stimulatory effect requires the presence of the proteins of the Phosphoenolpyruvate; sugar Phosphotransferase System. C. Involvement of Enzyme I of the Phosphoenolpyruvate: Sugar Phosphotransferase System as an Activator of Adenylyl Cyclase in E. coli. Enzyme IIIglc of the Phosphoenolpyruvate: sugar Phosphotransferase System has been concluded to be a positive regulator of adenylyl cyclase activity when it is in the phosphorylated form. Since Enzyme I of this phosphotransferase system is required to maintain Enzyme IIIglc in the phospho-form, it had been assumed that this protein acts only indirectly to activate adenylyl cyclase. A strategy was developed to allow Enzyme I to phosphorylate Enzyme IIIglc but not form a complex with the protein. Under these conditions, the phosphorylated form of Enzyme IIIglc did not stimulate adenylyl cyclase activity. The conclusion from these studies is that Enzyme I is actually an activator of adenylyl cyclase.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Heart, Lung, and Blood Institute (NHLBI)
• Type: Intramural Research (Z01)
• Project #: 1Z01HL000151-22
• Application #: 3843234
• Support Year: 22
• Fiscal Year: 1992

Institution

• Name: National Heart, Lung, and Blood Institute
• Country: United States

Publications

Wang, Guangshun; Peterkofsky, Alan; Keifer, Paul A et al. (2005) NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr. Protein Sci 14:1082-90

Williams Jr, David C; Cai, Mengli; Suh, Jeong-Yong et al. (2005) Solution NMR structure of the 48-kDa IIAMannose-HPr complex of the Escherichia coli mannose phosphotransferase system. J Biol Chem 280:20775-84

Koo, Byoung-Mo; Yoon, Mi-Jeong; Lee, Chang-Ro et al. (2004) A novel fermentation/respiration switch protein regulated by enzyme IIAGlc in Escherichia coli. J Biol Chem 279:31613-21

Keifer, Paul A; Peterkofsky, Alan; Wang, Guangshun (2004) Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide. Anal Biochem 331:33-9

Legler, Patricia M; Cai, Mengli; Peterkofsky, Alan et al. (2004) Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter IImannitol of the Escherichia coli phosphotransferase system. J Biol Chem 279:39115-21

Sondej, Melissa; Vazquez-Ibar, Jose Luis; Farshidi, Arta et al. (2003) Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand. Biochemistry 42:9153-9

Dimitrova, Mariana N; Peterkofsky, Alan; Ginsburg, Ann (2003) Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli. Protein Sci 12:2047-56

Wang, Guangshun; Keifer, Paul A; Peterkofsky, Alan (2003) Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles. Protein Sci 12:1087-96

Li, Xia; Peterkofsky, Alan; Wang, Guangshun (2003) 1H, 15N, and 13C chemical shift assignments of the Escherichia coli nitrogen regulatory phosphocarrier IIA(Ntr). J Biomol NMR 27:401-2

Reddy, Prasad T; Prasad, C Rama; Reddy, P Hemalatha et al. (2003) Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin. J Bacteriol 185:5263-8