title: Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.
abstract: Pyridoxal 5'-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6) proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6) enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
authors: Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors: Mohini S
authors: MS
authors: Ghatge
authors: Roberto
authors: R
authors: Contestabile
authors: Martino L
authors: ML
authors: di Salvo
authors: Jigar V
authors: JV
authors: Desai
authors: Amit K
authors: AK
authors: Gandhi
authors: Christina M
authors: CM
authors: Camara
authors: Rita
authors: R
authors: Florio
authors: Isabel N
authors: IN
authors: González
authors: Alessia
authors: A
authors: Parroni
authors: Verne
authors: V
authors: Schirch
authors: Martin K
authors: MK
authors: Safo
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011731:Pyridoxal Kinase