Abstract
Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.
Highlights
F’yridoxal kinase has been purified 2,000-fold from pig brain
Substrates of Pyridoxal Kinase- it is generally accepted that pyridoxal kinase phosphorylates all the nonphosphorylated vitamins, i.e. pyridoxal, pyridoxine, and pyridoxamine, it was thought worthwhile to investigate whether similar reactions take place in the presence of purified pyridoxal kinase
The catalytic activity of the kinase toward the substrate pyridoxine was studied by using an enzymatic assay system consisting of two purified enzymes, pyridoxal kinase and pyridoxine-5-P oxidase
Summary
F’yridoxal kinase has been purified 2,000-fold from pig brain. Riboflavin binds to the kinase with a Ko = 5 pM as shown by fluorometric titrations. It has been shown that the mechanisms by which brain slices concentrate kinase (3). Despite these studies which indicate the important role played by pyridoxal kinase in the metabolism and transport of vitamin Bs, little is known about the mechanism of action of this enzyme. No information is available about the amino acid residues responsible for binding the substrates and the amino acid residues which participate in the catalytic step. To study the specificity of the reaction catalyzed by purified pyridoxal kinase and secondly, to investigate the amino acid residues critically connected with enzyme activity
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