Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.
1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.
D000624:5-Aminolevulinate Synthetase / Q000378:metabolism*; D001894:Borohydrides / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D003486:Cyanides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism* / Q000494:pharmacology; D011733:Pyridoxamine / Q000494:pharmacology; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D001894:Borohydrides; D003486:Cyanides; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D000624:5-Aminolevulinate Synthetase