RESUMO
BACKGROUND: Defects in the enzyme coproporphyrinogen oxidase result in accumulation of porphyrins which may affect the severity of a subset of porphyrias. Thus evaluation of this enzyme for substrate selectivity is of value. Kinetic evaluations of recombinant human coproporphyrinogen oxidase have been undertaken using six di- and tripropionate analogs of the natural substrate coproporphyrinogen-III. These substrate analogs were modified by having alkyl groups in place of one or both of the ring 13- or 17-propionate moieties. MATERIAL/METHODS: Cloned human enzyme was incubated with analogs under apparent first order conditions and with various substrate concentrations. The kinetic values, K(m) and V(max), were determined. RESULTS: Relative to the authentic substrate, the K(m) values for the 13-ethyl, dimethyl and diethyl porphyrinogens were very comparable whereas the K(m) values were much higher using dipropyl and dibutyl porphyrinogen and much lower for the 17-ethyl analog. For the dipropionate analogs, the V(max) values were an apparent function of the carbon length of the substituent on the C and D rings, with longer carbon length severely reducing product formation by some 4-5 orders of magnitude. Also, the two isomeric tripropionates that were tested indicated that it was more detrimental to have an ethyl group at the 13-position for both binding and catalysis. CONCLUSIONS: This work extends our understanding of porphyrin ring substituent effects reported by Cooper et al. (2005). The substituents on both the C and D rings have significant effects on both the substrate binding and catalysis by this important enzyme.