INHIBITION OF HUMAN GLUTATHIONE-REDUCTASE BY S-NITROSOGLUTATHIONE
INHIBITION OF HUMAN GLUTATHIONE-REDUCTASE BY S-NITROSOGLUTATHIONE
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DOI：
10.1111/j.1432-1033.1995.472_b.x
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发表时间：
1995-12-01
期刊：
影响因子：
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通讯作者：
SCHIRMER, RH
中科院分区：
文献类型：
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作者：
BECKER, K;GUI, M;SCHIRMER, RH
亚硝基谷胱甘肽（GSNO）是一氧化氮（NO）在生物体内的主要转运形式。由于NO和GSNO已被证明可以调节各种蛋白质的功能，我们研究了GSNO和其他NO供体对人谷胱甘肽还原酶（GR）的影响。催化反应NADPH + GSSG + H+ -> NADP（+）+ 2 GSH，二聚体黄蛋白GR是谷胱甘肽氧化还原代谢的中心酶。GSNO被发现以两种方式抑制结晶红细胞GR：（a）作为可逆抑制剂GSNO与二硫代谷胱甘肽（GSSG）竞争，Ki约为0.5 mM;（B）作为不可逆抑制剂;与1 mMGSNO孵育1小时（3小时）后，GR（2.5U/ml，代表红细胞内浓度）被抑制70%（90%）。这种抑制依赖于NADPH的存在，并且不能通过稀释或还原剂逆转。吸收光谱表明，Cys 63和黄素之间的电荷转移相互作用被取消了这种修改。GSNO对GR抑制90%的样品中，底物GSSG和NADPH的Km值没有显著变化，修饰也不诱导酶的氧化酶活性。发现GSNO不是GR正向反应的底物。这意味着GSNO不能用GR测定总谷胱甘肽的方法来解释。即1 mM硝普钠或1 mM S-亚硝基-N-乙酰基-DL-青霉胺（SNAP）分别仅产生25%和10%的抑制。对于纯化的重组GR，即缺少包括Cys 2在内的15个N-末端氨基酸的GR突变体，以及对于稀释的新鲜溶血产物中存在的酶，获得了与纯化的真实GR相当的GSNO抑制模式（0.02 U/ml）;在浓缩的溶血产物中抑制作用不太明显，结果表明，GSNO对GR的不可逆抑制作用与酶催化位点Cys 63和/或Cys 58的亚硝基化有关。为了进一步研究失活的机制，我们结晶了GSNO修饰的GR用于X射线衍射分析。
S-Nitrosoglutathione (GSNO) represents a major transport form of nitric oxide (NO) in biological systems. Since NO and GSNO have been shown to modulate the function of various proteins, we studied the influence of GSNO and other NO donors on human glutathione reductase (GR). Catalyzing the reaction NADPH + GSSG + H+ --> NADP(+) + 2 GSH, the dimeric flavoprotein GR is the central enzyme of the glutathione redox metabolism.GSNO was found to inhibit crystalline erythrocyte GR in two ways: (a) as a reversible inhibitor GSNO is competitive with glutathione disulfide (GSSG), the K, being appr, 0.5 mM; (b) as an irreversible inhibitor; after 1 h (3 h) incubation with 1 mM GSNO, GR (2.5 U/ml, representing intraerythrocytic concentrations) was inhibited by 70% (90%). This inhibition depended on the presence of NADPH and could not be reversed by dilution nor by reducing agents. Absorption spectra indicate that the charge-transfer interaction between Cys63 and the flavin is abolished by this modification. In a GR sample inhibited by 90% with GSNO, the K-m values for the substrates GSSG and NADPH were not significantly changed nor did the modification induce oxidase activity of the enzyme.GSNO was found not to be a substrate in the forward reaction of GR. This implies that GSNO is not accounted for by methods which employ GR for determining total glutathione.Incubating isolated GR for 60 min with other NO donors, namely 1 mM sodium nitroprusside or 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP), resulted in only 25% and 10% inhibition, respectively. This attests to a specific affinity of GSNO to the enzyme.GSNO inhibition patterns comparable to purified authentic GR were obtained for purified recombinant GR, a GR mutant lacking the 15 N-terminal amino acids including Cys2, and for the enzyme present in diluted fresh haemolysates (0.02 U/ml); in concentrated haemolysates the inhibition was less pronounced, GR of intact erythrocytes was not affected when exposed to GSNO in the medium.Our results suggest that the irreversible inhibition of GR by GSNO involves nitrosylation of Cys63 and/or Cys58 at the catalytic site of the enzyme. To further investigate the mechanism of inactivation we have crystallized GSNO-modified GR for X-ray diffraction analysis.