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. © 2004 Elsevier Inc. All rights reserved.

Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases / DI SALVO, Martino Luigi; Sharyn, Hunt; Verne, Schirch. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - 36:2(2004), pp. 300-306. [10.1016/j.pep.2004.04.021]

Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases

DI SALVO, Martino Luigi;
2004

Abstract

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. © 2004 Elsevier Inc. All rights reserved.
2004
pyridoxal kinase; pyridoxal phosphate
01 Pubblicazione su rivista::01a Articolo in rivista
Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases / DI SALVO, Martino Luigi; Sharyn, Hunt; Verne, Schirch. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - 36:2(2004), pp. 300-306. [10.1016/j.pep.2004.04.021]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/127856
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