Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP+ binding affects both folding and stability
Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP+ binding affects both folding and stability
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DOI：
10.1016/j.bbadis.2009.05.003
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发表时间：
2009-08-01
影响因子：
6.2
通讯作者：
Engel, Paul C.
中科院分区：
文献类型：
--
作者：
Wang, Xiao-Tao;Engel, Paul C.
人葡萄糖6-磷酸脱氢酶（G6 PD）具有“催化性”NADP（+）位点和“结构性”NADP（+）位点，其中许多严重的G6 PD缺陷突变位于该位点。两对G6 PD临床突变体（威斯康星州）（R393 G）和G6 PD（纳什维尔）（R393 H）、G6 PD（福谷）（G488 S）和G6 PD（Campims）（G488 V），其中突变位于“结构”NADP（+）位点附近，显示“结构”NADP（+）的K-d值升高，范围为53 nM至500 nM，而野生型酶为37 nM。这些重组酶用Gdn-HCl变性，并在L-Arg、NADP（+）和DTT存在下于25 ℃通过快速稀释进行复性。突变体的重折叠产率表现出强烈的NADP（+）依赖性，在1000 pM NADP（+）的范围从1.5%到59.4%，在所有情况下低于野生型酶的72%的数字。这些突变酶也表现出降低的热稳定性和高敏感性糜蛋白酶消化，在CD实验中与它们相应的熔化温度良好的协议。总之，结果支持这样的观点，即受损的“结构”NADP（+）的结合可以阻碍折叠以及导致这些临床突变酶在完全折叠状态下的不稳定性。(C)2009爱思唯尔有限公司版权所有。
Human glucose 6-phosphate dehydrogenase (G6PD) has both the "catalytic" NADP(+) site and a "structural" NADP(+) site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD(Wisconsin) (R393G) and G6PD(Nashville) (R393H), and G6PD(Fukaya) (G488S) and G6PD(Campims) (G488V), in which the mutations are in the vicinity of the "structural" NADP(+) site, showed elevated K-d values of the "structural" NADP(+), ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of L-Arg, NADP(+) and DTT at 25 degrees C. The refolding yields of the mutants exhibited strong NADP(+)-dependence and ranged from 1.5% to 59.4% with 1000 pM NADP(+), in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of "structural" NADP(+) can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state. (C) 2009 Elsevier B.V. All rights reserved.