Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases.
Pyridoxal kinase is an ATP dependent enzyme that phosphorylates pyridoxal, pyridoxine, and pyridoxamine forming their respective 5'-phosphorylated esters. The kinase is a part of the salvage pathway for re-utilizing pyridoxal 5'-phosphate, which serves as a coenzyme for dozens of enzymes involved in amino acid and sugar metabolism. Clones of two pyridoxal kinases from Escherichia coli and one from human were inserted into a pET 22b plasmid and expressed in E. coli. All three enzymes were purified to near homogeneity and kinetic constants were determined for the three vitamin substrates. Previous studies had suggested that ZnATP was the preferred trinucleotide substrate, but our studies show that under physiological conditions MgATP is the preferred substrate. One of the two E. coli kinases has very low activity for pyridoxal, pyridoxine, and pyridoxamine. We conclude that in vivo this kinase may have an alternate substrate involved in another metabolic pathway and that pyridoxal has only a poor secondary activity for this kinase.
di Salvo
Martino L
ML
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università degli Studi di Roma, La Sapienza, Rome, Italy.
Hunt
Sharyn
S
Schirch
Verne
V
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D050260:Carbohydrate Metabolism; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008660:Metabolism / Q000502:physiology; D010957:Plasmids; D011731:Pyridoxal Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity / Q000502:physiology; D025101:Vitamin B 6 / Q000378:metabolism
D000596:Amino Acids; D003067:Coenzymes; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D025101:Vitamin B 6; D000255:Adenosine Triphosphate; D011731:Pyridoxal Kinase