旧黄色酵素の活性域について
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概要
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For the understanding of the molecular basis of the flavoenzyme catalyses in general terms, studies on old yellow enzyme (OYE) have been carried out from various points of view. We have found the microheterogeneity of OYE and separated the enzyme, which had been treated as a single entity, into five different subforms, OYE-I, II, III, IV and V, by means of anion exchange HPLC. These subforms exhibited similar NADPH oxidase activities but distinct microenvironments surrounding FMN as revealed by ^<13>C NMR with ^<13>C-enriched FMN. The limited proteolysis of OYE with α-chymotrypsin cleaved at a specific site yielding two fragments of molecular weights of 14,000 and 34,000. These fragments are tightly associated with each other forming a complex termed "nicked OYE" except under stringent denaturating conditions. Nicked OYE still retained FMN as the coenzyme and exhibited slightly modified kinetic and spectral properties as compared to native OYE. Covalently-flavinylated OYE was obtained by a combination of the limited proteolysis with α-chymotrypsin and reconstitution of OYE with 8-fluoro-8-demethyl-FMN (8F-FMN). The flavinylation took place between the amino group of the N-terminal leucine of the 14K fragment and the 8-position of the 8F-FMN. That the flavinylation took place within the 14K fragment indicated that in native OYE the 14K domain is the FMN binding domain. The amino acid sequence around the flavinylation site shows homology with a certain stretch of sequence in flavodoxin arguing in favor of the 14K domain being the FMN binding domain.
- 日本ビタミン学会の論文
- 1990-10-25
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