title: Deuteration of Escherichia coli enzyme I(Ntr) alters its stability.
abstract: Enzyme I(Ntr) is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI(Ntr). Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4°C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI(Ntr) are evaluated.
authors.affiliation: The National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Lee
authors.forename: Nico
authors.initials: N
authors.lastname: Tjandra
authors.forename: Chang-Ro
authors.initials: CR
authors.lastname: Lee
authors.forename: Yeong-Jae
authors.initials: YJ
authors.lastname: Seok
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
authors.forename: Alan
authors.initials: A
authors.lastname: Peterkofsky
mesh_terms: D000595:Amino Acid Sequence; D001119:Arginase / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D028044:Phosphate-Binding Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D058767:Protein Unfolding; D013696:Temperature; D051844:alpha-Synuclein / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; C502371:NPr protein, E coli; D028044:Phosphate-Binding Proteins; D051844:alpha-Synuclein; D003903:Deuterium; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D001119:Arginase