Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate.
Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate.
复制标题
焦碳酸二乙酯灭活干酪乳杆菌的二氢叶酸还原酶。
作者：
H. Daron;J. Aull
用二酸乳杆菌的二氢叶酸还原酶的组氨酸残基与二乙基氯苯甲酸二乙酯进行了研究。该酶没有半胱氨酸残基，并且在许多烟酰胺核苷酸脱氢酶中有所不同，这些氧化酰胺核苷酸脱氢酶具有催化重要的硫酸基团。对该酶的X射线研究表明，组氨酸残基参与底物结合，但不参与质子转移[Matthews等。 （1978）J。Biol。化学253，6946]。二氢叶酸还原酶被二乙酯二乙二醇灭活；反应的二阶速率常数在0度C处为29 m-1 min-1。天然和硫化二乙基二苯甲酸二苯甲酸二氧化酶的差异最大接近242 nm，这表明与组氨酸残基有反应。在280 nm附近没有任何光谱差异表明二乙基碳酸盐盐未与酪氨酸残基反应。二氢叶酸还原酶在修改了七个组氨酸残基中约有六个后失去了所有酶促活性。在与大约四个组氨酸残基的初始快速反应中，催化活性均未丢失，但是随后较慢的反应涉及另外一个或两个残基与活性丧失有关。酶受到NADPH或二氢叶酸的底物的灭活保护。实际上，在两种底物存在下，尤其是NADPH的情况下，用二乙基碳酸二碳酸盐处理的活性大大比未经处理的酶发现的活性要大得多。用1 M羟胺处理对二氢叶酸还原酶的活性部分恢复，而二乙基乙酸二乙酯灭活。
The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al. (1978) J. Biol. Chem. 253, 6946]. Dihydrofolate reductase was inactivated by diethyl pyrocarbonate; the second-order rate constant for the reaction was 29 M-1 min-1 at 0 degrees C. The difference spectrum of native and diethyl pyrocarbonate inactivated enzyme had a maximum near 242 nm, which indicated a reaction with histidine residues. The absence of any spectral difference near 280 nm indicated that diethyl pyrocarbonate had not reacted with tyrosine residues. Dihydrofolate reductase lost all of its enzymatic activity after about six of the seven histidine residues had been modified. No catalytic activity was lost during an initial rapid reaction with about four histidine residues, but a subsequent slower reaction involving an additional one or two residues was associated with the loss of activity. The enzyme was protected from inactivation by either of the substrates NADPH or dihydrofolate. In fact, treatment with diethyl pyrocarbonate in the presence of either substrate, but particularly with NADPH, resulted in substantially greater activity than that found with untreated enzyme. Treatment with 1 M hydroxylamine partially restored activity to dihydrofolate reductase that had been inactivated by diethyl pyrocarbonate.