Studies on the Active Site of Brevibacterium Pyrocatechase

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Abstract
Brevibacterium pyrocatechase [EC 1.13.1.1] contains a bound ferric iron essential for its enzymic activity. This bound ferric iron plays a role in the enzymic reaction at both the catalytic and substrate binding sites. A sulfhydryl group of apopyrocatechase (iron-free) reacts with 5,5′-dithiobis(2-nitro-benzoic acid) (DTNB) resulting in the formation of the TNB-bound apoenzyme (TNB-apoenzyme) which has no ability to bind iron. By reacting this with cysteine, to reduce the disulfide bond, the reconstitution of holopyrocatechase (iron-containing) is achieved and full enzymic activity restored. Both native and reconstituted holo-enzyme have no DTNB-detectable sulfhydryl group. The holoenzyme as well as the apoenzyme reacts with one molar equivalent of pchloromercuribenzoate (PCMB) and completely loses enzymic activity. These observations indicate the existence of an iron-sulfur linkage in pyrocatechase. By chemical modification using N-bromosuccinimide (NBS) together with fluoro-metric studies, the participation of a tryptophanyl residue in the binding of iron to pyrocatechase was strongly suggested. On treatment of a single tryptophanyl residue in apopyrocatechase with small amount of NBS, the ability to recombine an iron atom is lost. In the holoenzyme, this tryptophanyl residue is protected from NBS reaction. Both the holo- and apoenzyme showed a fluorescence emission maximum at 342 nm depending on tryptophanyl residue in the protein moiety after excitation at 280 nm. The emission intensity at 342 nm was 3-fold higher for the apoenzyme. The de-crease in fluorescence intensity of the apoenzyme accompanied by the addition of iron was in proportion to the binding of iron to the apoenzyme and vice versa. The structure of the active site of pyrocatechase is discussed on the basis of the above results.

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