01 November 2005
Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate
Christopher L. Cooper, Timothy D. Lash, Marjorie A. JonesMed Sci Monit 2005; 11(11): BR420-425 :: ID: 430299
Abstract
Background: The enzyme coproporphyrinogen oxidase (copro’gen oxidase) converts coproporphyrinogen-III
(C-III) 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 purifi ed human copro’gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs.
Materials/Methods: Therefore, purifi ed, cloned human copro’gen oxidase was incubated with C-III or C-IV at 37oC with various substrate concentrations (from 0.005 µM to 3.5 µM). 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 effi ciency (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.
Keywords: Coproporphyrinogen Oxidase - genetics, Cloning, Molecular, Catalysis, Coproporphyrinogens - chemistry, Isomerism, Kinetics, Substrate Specificity
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