Distinctive iron requirement of tryptophan 5-monooxygenase: TPH1 requires dissociable ferrous iron.
A peripheral type of tryptophan 5-monooxygenase (EC 1.14.16.4), TPH1, is very unstable in vitro, but the inactivation was reversible and full reactivation occurs upon anaerobic incubation with a high concentration of dithiothreitol (DTT, 15 mM). In this study, distinctive iron requirement of TPH1 was revealed through analysis of the enzyme's inactivation and activation by DTT. For this purpose, all the glasswares, plastics, Sephadex G-25 gels, and reagents including protein solutions had been treated with metal chelators, and apo-TPH was prepared by treatment with EDTA. Apo-TPH thus prepared exclusively required free Fe2+ for its catalytic activity; 10(-8) M was enough under the strict absence of Fe3+ but 10(-12) M was too low. No other metal ions including Fe3+ were effective. It appeared that Fe3+ bound to the enzyme with a higher affinity than Fe2+, resulting in the inactivation. Ascorbate, a non-thiol reducing agent, did not substitute DTT in the activation of TPH1, but enhanced the Fe2+-dependent activity of apo-TPH as effectively as DTT. Thus, the DTT-activation was essentially substituted by preparation of apo-TPH by the EDTA treatment and the assay of apo-TPH in the presence of Fe2+ and ascorbate. The activation of TPH1 by incubation with DTT was accompanied by exposure of 9 sulfhydryls out of the total 10 cysteine residues, but the cleavage of disulfide bonds seemed not to be crucial, even if it occurred. The effect of DTT was substituted by some other sulfhydryls whose structure was analogous to that of commonly used metal chelators. Based on these observations, the following dual roles of DTT are proposed: (1) in the activation of TPH, DTT removes inappropriate bound iron (Fe3+) as a chelator, keeping Fe3+ away from the enzyme's binding site which needs to bind Fe2+ for the catalytic activity, and (2) in both the activation and reaction processes, DTT prevents oxidation of Fe2+ to Fe3+ as a reducing agent.
Hasegawa
Hiroyuki
H
Department of Biosciences, Teikyo University of Science and Technology, Uenohara, Yamanashi 409-0193, Japan. hasegawa@ntu.ac.jp
Ichiyama
Arata
A
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005260:Female; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008297:Male; D034801:Mastocytoma / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000737:chemistry / Q000378:metabolism*
D005296:Ferrous Compounds; D008024:Ligands; D013438:Sulfhydryl Compounds; C487801:Tph1 protein, mouse; D014365:Tryptophan Hydroxylase