Abstract
Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B6 requiring enzymes. PLP reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B6 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-B6 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-B6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
Highlights
Pyridoxal 59-phosphate (PLP) is a cofactor for dozens of enzymes in the E. coli cell that are important in amino acid metabolism, as well as in several other pathways [1]
We report here on properties of E. coli PL kinase showing that PLP serves as a slow tight binding inhibitor of the enzyme
Inactivation of ePL Kinase during Catalytic Turnover During our assay of ePL kinase to determine kinetic constants we observed a rapid loss of activity making kinetic studies from initial rates difficult
Summary
Pyridoxal 59-phosphate (PLP) is a cofactor for dozens of enzymes in the E. coli cell that are important in amino acid metabolism, as well as in several other pathways [1]. The regulation of PLP homeostasis in E. coli and how each of the dozens of PLP requiring apo-B6 enzymes competes for available PLP, to form the catalytically active holo-B6 enzymes, are important unresolved problems. The biosynthetic pathway results in the formation of pyridoxine 59-phosphate (PNP, reaction 1), which is converted to PLP by the flavin enzyme PNP oxidase (reaction 2) [3]. PLP adds to newly synthesized apo-B6 enzymes converting them to the catalytically active holo enzymes (reaction 3). PLP is released during protein turnover of B6 enzymes (reaction 4) and is converted to pyridoxal (PL) by cellular phosphatases (reaction 5) [3,4,5]. PL kinase, PNP oxidase and PLP phosphatase constitute the salvage pathway
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