Catechol dioxygenases substrate activation

Figure 6 Proposed substrate activation mechanism for intradiol-cleaving catechol dioxygenases. Substrate activation is proposed to occur via an iron(II)-semiqumone prior to O2 binding...

There are many discussions on the coordination numbers of the iron species in the resting and intermediate states. It is noteworthy that in many cases a six coordination is first proposed, but importance of five coordination is demonstrated. As seen in catechol dioxygenase, substrates coordinate to iron with displacing attached ligands such as histidine with forming five coordinate species. The unsaturated coordination around the iron center may be important for the oxygen activation and oxygenation. 

An enzymatic pathway for indole degradation was found in A. niger, inducible by the substrate within a 5-h period during growth. Among the enzymes found, anthranilate hydroxylase, N-formylanthranilate deformylase, 2,3-dihydroxybenzoate decarboxylase, and catechol dioxygenase were isolated, and their activities were demonstrated in a cell-free system [342],... 

Figure 15 Proposed substrate activation mechanism for the intradiol cleaving catechol dioxygenases.

Coordination of the active site metal by water molecules and substrates is obviously much more variable and the X-ray structure may be of little assistance. In order to determine experimentally the orientation of the substrate, a crystal would need to be grown with the substrate bound. Notwithstanding the problems of obtaining X-ray quality crystals in the first place, by its very nature, the enzyme will attempt to convert the substrate to product and capturing a bound state may not be possible. However, many reactions require an additional reactant—say a molecule of O2—and thus the substrate-bound form may remain stable under anaerobic conditions. The structure of catechol dioxygenase with substrate bound has been determined in this way [36]. 

Formation of a substrate-metal binding, that is important for activation of substrate, has been suggested from the formation of stable model substrate-metal complexes. Recently, however, the substrate-bound iron enzymes have been demonstrated by X-ray crystallography in the cases of intradiol [15] and extradiol [9] catechol dioxygenases It is possible that these species isolated is not the direct intermediate involved in the catalytic cycle but those stabilized in the isolation process, but crystallographic data provide strong supports for the substrate-metal species. 

Importance of the ferrous state for dioxygenation is mostly observed, but intradiol catechol dioxygenases and lipoxygenase require the ferric state for oxygenation. The oxidation state of iron important for oxygenation is sometimes different from that of isolated enzymes and in the resting state. In the enzymes which require the ferric state, activation of substrate by the iron center seems to proceed in preference to that of oxygen, and vice versa in the ferrous enzymes. 

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