Li Tingting, Woods James S
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105, USA.
Toxicol Sci. 2009 Jun;109(2):228-36. doi: 10.1093/toxsci/kfp066. Epub 2009 Apr 1.
Coproporphyrinogen oxidase (CPOX) catalyzes the two-step decarboxylation of coproporphyrinogen-III to protoporphyrinogen-IX in the heme biosynthetic pathway. Previously we described a specific polymorphism (A814C) in exon 4 of the human CPOX gene (CPOX4) and demonstrated that CPOX4 is associated with both modified urinary porphyrin excretion and increased neurobehavioral deficits among human subjects with low-level mercury (Hg) exposure. Here, we sought to characterize the gene products of CPOX and CPOX4 with respect to biochemical and kinetic properties. Coproporphyrinogen-III was incubated with recombinantly expressed and purified human CPOX and CPOX4 enzymes at various substrate concentrations, with or without Hg(2+) present. Both CPOX and CPOX4 formed protoporphyrinogen-IX from coproporphyrinogen-III; however, the affinity of CPOX4 was twofold lower than that of CPOX (CPOX K(m) = 0.30 microM, V(max) = 0.52 pmol protoporphyrin-IX; CPOX4 K(m) = 0.54 microM, V(max) = 0.33 pmol protoporphyrin-IX). Hg(2+) specifically inhibited the second step of coproporphyrinogen-III decarboxylation (harderoporphyrinogen to protoporphyrinogen-IX) in a dose dependent manner. We also compared the catalytic activities of CPOX and CPOX4 in human liver samples. The specific activities of CPOX in mutant livers were significantly lower (40-50%) than those of either wild-type or heterozygous. Additionally, enzymes from mutant, heterozygous and wild-type livers were comparably inhibited by Hg(2+) (10 microM), decreasing CPOX4 activity to 25% that of the wild-type enzyme. These findings suggest that CPOX4 may predispose to impaired heme biosynthesis, which is limited further by Hg exposure. These effects may underlie increased susceptibility to neurological deficits previously observed in Hg-exposed humans with CPOX4.
粪卟啉原氧化酶（CPOX）在血红素生物合成途径中催化粪卟啉原III两步脱羧生成原卟啉原IX。此前我们描述了人类CPOX基因（CPOX4）外显子4中的一种特异性多态性（A814C），并证明CPOX4与低水平汞（Hg）暴露人群中尿卟啉排泄改变及神经行为缺陷增加有关。在此，我们试图从生化和动力学特性方面对CPOX和CPOX4的基因产物进行表征。将粪卟啉原III与重组表达并纯化的人CPOX和CPOX4酶在不同底物浓度下孵育，有无Hg(2+)存在。CPOX和CPOX4均能将粪卟啉原III转化为原卟啉原IX；然而，CPOX4的亲和力比CPOX低两倍（CPOX K(m)=0.30 microM，V(max)=0.52 pmol原卟啉IX；CPOX4 K(m)=0.54 microM，V(max)=0.33 pmol原卟啉IX）。Hg(2+)以剂量依赖方式特异性抑制粪卟啉原III脱羧的第二步（硬卟啉原转化为原卟啉原IX）。我们还比较了人肝脏样本中CPOX和CPOX4的催化活性。突变肝脏中CPOX的比活性显著低于野生型或杂合子（低40 - 50%）。此外，来自突变、杂合和野生型肝脏的酶受到Hg(2+)（10 microM）的同等抑制，使CPOX4活性降至野生型酶的25%。这些发现表明CPOX4可能易导致血红素生物合成受损，而Hg暴露会进一步限制其合成。这些效应可能是先前在CPOX4基因的Hg暴露人群中观察到的神经缺陷易感性增加的基础。
