Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate.
BACKGROUND: The enzyme coproporphyrinogen oxidase (copro'gen oxidase) converts coproporphyrinogen-III (GIII) to protoporphyrinogen-IX via an intermediary monovinyl porphyrinogen. The A ring isomer coproporphyrinogen-IV (C-IV) has previously been shown to be a substrate for copro'gen oxidase derived from avian erythrocytes. In contrast to the authentic substrate (C-III) where only a small amount of the monovinyl intermediate is detected, C-IV gives rise to a monovinyl intermediate that accumulates before being converted to an isomer of protoporphyrinogen-IX. No kinetic studies have been carried out using the purified human copro'gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs. MATERIALS/METHODS: Therefore, purified, cloned human copro'gen oxidase was incubated with C-III or C-IV at 37 degrees C with various substrate concentrations (from 0.005 pM to 3.5 pM). The Km (an indication of molecular recognition) and Kcat (turnover number) values were determined. RESULTS: The Km value for total product formation was about the same with either C-III or C-IV indicating the same molecular recognition. However, the catalytic efficiency (Kcat/Km) of the enzyme for total product formation was not more than two fold higher using C-III relative to C-IV. CONCLUSIONS: Since the Km values are about the same for either substrate and the total Kcat/Km values are within two fold of each other, this could correlate with the increase of severity of porphyrias with monovinyl accumulation. The ability of the increased levels of C-IV to compete with the authentic substrate has important implications for clinical porphyrias.[1]References
- Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate. Cooper, C.L., Lash, T.D., Jones, M.A. Med. Sci. Monit. (2005) [Pubmed]
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