Occurrence of a novel high molecular weight activator of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides

• Published in: Journal of biochemistry (Tokyo) 1979-01, Vol.86 (2), p.483-489
• Main Authors: OYAMA, Hideko, TUBOI, Syozo
• Format: Article
• Language: English
• Subjects: 5-Aminolevulinate Synthetase - metabolism; Bacterial Proteins - isolation & purification; Bacterial Proteins - pharmacology; Cystathionine gamma-Lyase - metabolism; Cystine - pharmacology; Enzyme Activation; Molecular Weight; Rhodobacter sphaeroides - enzymology
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a132548

It was found that, in addition to the activating enzyme (cystathionase) and L-cystine, a novel high molecular weight activator (the regulator protein) is required for the activation of the inactive form of δ-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas spheroides. This regulator protein was purified from the cell extract to electrophoretic homogeneity. The regulator protein was converted by incubation with cystathionase and L-cystine to an active regulator protein which could activate the inactive form of ALA synthetase in the absence of cystathionase and L-cystine. When the regulator protein was incubated with 35S-cystine or 14C-cystine in the presence of cystathionase, it incorporated 35S, but not 14C, showing that only the S atom was incorporated from L-cystine into the regulator protein molecule by the action of cystathionase [EC 4.4.1.1]. The 35S-labeled regulator protein was digested by pronase (nonspecific proteinase), and a 35S-labeled compound obtained in the hydrolysate was identified as cystine trisulfide by Dowex 50 column chromatography and high-voltage paper electrophoresis. It was assumed, therefore, that the 35S atom of 35S-cystine is incorporated to form a cystine trisulfide structure with two cysteine residues in the regulator protein molecule. Based on these observations, the mechanism of activation of the inactive form of ALA synthetase is proposed to be as follows: at first the regulator protein is modified by cystathionase and L-cystine, forming a cystine trisulfide structure from the S-atom of cystine and two cysteine residues in the protein molecule; next, the modified regulator protein converts the inactive form of ALA synthetase to the active form by intramolecular thiol-disulfide interchange.