Becker K, Gui M, Schirmer R H
Institute of Biochemistry II, Heidelberg University, Germany.
Eur J Biochem. 1995 Dec 1;234(2):472-8. doi: 10.1111/j.1432-1033.1995.472_b.x.
S-Nitrosoglutathione (GSNO) represents a major transport form in 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 Ki 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 Km 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.
S-亚硝基谷胱甘肽（GSNO）是生物系统中一氧化氮（NO）的主要运输形式。由于NO和GSNO已被证明可调节多种蛋白质的功能，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。二聚体黄素蛋白GR催化反应NADPH + GSSG + H(+) --> NADP(+) + 2 GSH，是谷胱甘肽氧化还原代谢的核心酶。发现GSNO以两种方式抑制结晶红细胞GR：（a）作为可逆抑制剂，GSNO与谷胱甘肽二硫化物（GSSG）竞争，其Ki约为0.5 mM；（b）作为不可逆抑制剂；与1 mM GSNO孵育1小时（3小时）后，GR（2.5 U/ml，代表红细胞内浓度）被抑制70%（90%）。这种抑制取决于NADPH的存在，不能通过稀释或还原剂逆转。吸收光谱表明，这种修饰消除了Cys63与黄素之间的电荷转移相互作用。在被GSNO抑制90%的GR样品中，底物GSSG和NADPH的Km值没有显著变化，这种修饰也没有诱导该酶的氧化酶活性。发现GSNO不是GR正向反应的底物。这意味着采用GR测定总谷胱甘肽的方法无法检测到GSNO。将分离的GR与其他NO供体，即1 mM硝普钠或1 mM S-亚硝基-N-乙酰-DL-青霉胺（SNAP）孵育60分钟，分别仅导致25%和10%的抑制。这证明了GSNO对该酶具有特异性亲和力。对于纯化的重组GR、缺乏包括Cys2在内的15个N端氨基酸的GR突变体以及稀释的新鲜溶血产物（0.02 U/ml）中存在的酶，获得了与纯化的天然GR相当的GSNO抑制模式；在浓缩的溶血产物中，抑制作用不太明显。完整红细胞的GR在培养基中暴露于GSNO时不受影响。我们的结果表明，GSNO对GR的不可逆抑制涉及该酶催化位点的Cys63和/或Cys58的亚硝基化。为了进一步研究失活机制，我们已将GSNO修饰的GR结晶用于X射线衍射分析。
