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.


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.
Cooper
Christopher L
CL
Department of Chemistry, Illinois State University, Normal, IL 61790-4160, USA.
Lash
Timothy D
TD
Jones
Marjorie A
MA
D002384:Catalysis; D003001:Cloning, Molecular; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry* / Q000235:genetics; D003305:Coproporphyrinogens / Q000737:chemistry*; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D013379:Substrate Specificity
D003305:Coproporphyrinogens; C508176:coproporphyrinogen IV; C028026:coproporphyrinogen III; D003304:Coproporphyrinogen Oxidase