Kinetic properties of polymorphic variants and pathogenic mutants in human cystathionine γ-lyase
Kinetic properties of polymorphic variants and pathogenic mutants in human cystathionine γ-lyase
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DOI：
10.1021/bi800351a
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发表时间：
2008-06-10
期刊：
影响因子：
2.9
通讯作者：
Banerjee, Ruma
中科院分区：
文献类型：
--
作者：
Zhu, Weidong;Lin, Alexander;Banerjee, Ruma
人半胱硫氨酸- γ -裂解酶（CGL）是一种吡哆醛-5'-磷酸（PLP）依赖性酶，在将同型半胱氨酸转化为半胱氨酸的转硫途径中起作用。此外，CGL是可以催化硫化氢形成的两种主要酶之一，硫化氢是一种重要的气体信号分子。最近，在胱氨酸硫氨酸尿患者中发现了几种CGL突变，胱氨酸硫氨酸尿是一种罕见但知之甚少的遗传疾病。此外，CGL中常见的单核苷酸多态性c. 1364G >t可将403位的丝氨酸转化为异亮氨酸，这与血浆同型半胱氨酸水平升高有关。在本研究中，我们用动力学和分光光度法表征了致病性T67I和Q240E错义突变和氨基酸残基403的多态性变异。我们报道，多态性不影响酶的辅因子含量或其稳态动力学性质。与野生型CGL相比，T67I突变体的V-max降低了3.5倍，而Q240E突变体的V-max降低了70倍。两种致病突变体对半胱甘氨酸的K(M)s与野生型CGL相当。T67I和Q240E突变体的PLP含量分别比野生型酶低约4倍和80倍。用PLP对T67I突变体进行预孵育，使其活性恢复到野生型水平，而同样的处理只使Q240E突变体的活性部分恢复。这些结果表明，这两种突变都削弱了对PLP的亲和力，并表明具有这些突变的胱硫钠尿酸患者应该对吡哆醇治疗有反应。
Human cystathionine-gamma-lyase (CGL) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme, which functions in the transsulfuration pathway that converts homocysteine to cysteine. In addition, CGL is one of two major enzymes that can catalyze the formation of hydrogen sulfide, an important gaseous signaling molecule. Recently, several mutations in CGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Moreover, a common single nucleotide polymorphism in CGL, c. 1364G>T that converts serine at position 403 to isoleucine, has been linked to elevated plasma homocysteine levels. In this study, we have characterized the pathogenic T67I and Q240E missense mutations and the polymorphic variants at amino acid residues 403 using kinetic and spectrophotometric methods. We report that the polymorphism does not influence the cofactor content of the enzyme or its steady-state kinetic properties. In contrast, the T67I mutant exhibits a 3.5-fold decrease in V-max compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in V-max. The K(M)s for cystathionine for both pathogenic mutants are comparable to that of wild type CGL. The PLP content of the T67I and Q240E mutants were about 4-fold and 80-fold lower than that of wild-type enzyme, respectively. Preincubation of the T67I mutant with PLP restored activity to wild-type levels while the same treatment resulted in only partial restoration of activity of the Q240E mutant. These results reveal that both mutations weaken the affinity for PLP and suggest that cystathionuric patients with these mutations should be responsive to pyridoxine therapy.