Morgenthaler Justin B, Barto Reyna L, Lash Timothy D, Jones Marjorie A
Department of Chemistry, Illinois State University Normal, IL 61790-4160, USA.
Med Sci Monit. 2008 Jan;14(1):BR1-7.
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.
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.
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.
粪卟啉原氧化酶缺陷会导致卟啉积累，这可能会影响部分卟啉病的严重程度。因此，评估该酶的底物选择性具有重要意义。已使用天然底物粪卟啉原III的六种二丙酸酯和三丙酸酯类似物对重组人粪卟啉原氧化酶进行了动力学评估。这些底物类似物通过用烷基取代环13-或17-丙酸酯部分中的一个或两个进行了修饰。
材料/方法：将克隆的人酶在表观一级条件下与类似物以及不同底物浓度一起孵育。测定动力学值K(m)和V(max)。
相对于真实底物，13-乙基、二甲基和二乙基卟啉原的K(m)值非常相近，而使用二丙基和二丁基卟啉原时K(m)值要高得多，17-乙基类似物的K(m)值则低得多。对于二丙酸酯类似物，V(max)值显然是C环和D环上取代基碳链长度的函数，碳链越长，产物形成减少约4-5个数量级。此外，测试的两种异构三丙酸酯表明，在13位有一个乙基对结合和催化都更不利。
这项工作扩展了我们对库珀等人（2005年）报道的卟啉环取代基效应的理解。C环和D环上的取代基对这种重要酶的底物结合和催化都有显著影响。
