Suzuki K, Mizuguchi M, Gomi T, Itagaki E
Department of Chemistry, Faculty of Science, Kanazawa University, Ishikawa.
J Biochem. 1995 Mar;117(3):579-85. doi: 10.1093/oxfordjournals.jbchem.a124747.
Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.
恶臭假单胞菌S-1中的水杨酸羟化酶被三硝基苯磺酸（TNBS）不可逆地失活。该反应与TNBS浓度呈线性相关，在pH 8.5时，全酶的二级反应速率常数为120 M-1·min-1。每摩尔酶修饰一摩尔赖氨酸残基会导致活性大幅丧失，且解偶联后该酶不再具有NADH脱氢酶活性。NADH、NAD+、ATP或AMP的存在可提供对失活的保护。通过疏水色谱法分离出在单个赖氨酸残基处修饰的酶，呈脱辅基蛋白形式并进行了表征。它结合FAD的解离常数（Kd）与天然脱辅基蛋白相同。该酶对NADH的表观米氏常数增加了13倍，但对水杨酸的表观米氏常数未变。NADH氧化的最大反应速度（Vmax）降至天然酶的五分之一。从修饰酶的胰凝乳蛋白酶消化物中分离出一个含有一个三硝基苯基赖氨酸残基的肽段，并确定其氨基酸序列为TADVAIAADGIKSSM，该序列与恶臭假单胞菌PpG7的水杨酸羟化酶从R-154到I-168的序列同源。该肽段中的赖氨酸可能代表一个与酶结合位点中NADH的阴离子基团相互作用的碱性残基。
