Hackett C S, Novoa W B, Kensil C R, Strittmatter P
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
J Biol Chem. 1988 Jun 5;263(16):7539-43.
Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.
在细胞色素b5还原酶可溶性催化片段中，烟酰胺腺嘌呤二核苷酸（NADH）结合位点外的赖氨酸残基用乙基亚氨酸乙酯和乙酸酐进行修饰，同时通过形成稳定的氧化核苷酸-还原黄素蛋白复合物来保护结合位点。这种处理对酶活性的影响最小；天然还原酶中与铁氰化钾反应的周转数为45,300，衍生物中为39,200。去除NADH后，随后与[3H]乙酸酐反应导致91%的酶活性丧失，并且有1.9当量的乙酰基掺入蛋白质中。在pH 13下用1 M羟胺处理表明只有赖氨酸残基被乙酰化，用溴化氰对衍生物进行片段化以及用胰蛋白酶和糜蛋白酶进行亚片段化表明，只有Lys110以高比活性被标记，化学计量比为0.83个乙酰基/摩尔，这与观察到的酶活性丧失情况非常吻合。其余的标记物在四个或更多其他赖氨酸残基中以低水平分布。这些结果表明，只有Lys110受到NADH的特异性保护，因此是在细胞色素b5还原酶中提供参与NADH结合的ε-氨基的残基。
