Renosto F, Schultz T, Re E, Mazer J, Chandler C J, Barron A, Segel I H
J Bacteriol. 1985 Nov;164(2):674-83. doi: 10.1128/jb.164.2.674-683.1985.
ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.
产黄青霉（一种嗜温菌）和杜邦青霉（一种嗜热菌）的ATP硫酸化酶的天然分子量约为440,000，亚基分子量约为69,000。（杜邦青霉的亚基似乎比产黄青霉的亚基略小。）杜邦青霉的酶比产黄青霉的酶热稳定性高约100倍；在65℃时，杜邦青霉的失活常数k（一级失活速率常数）= 3.3×10⁻⁴ s⁻¹，产黄青霉的为3.0×10⁻² s⁻¹。杜邦青霉的酶在30℃时对低pH和尿素也更稳定。针对每种酶的兔血清抗体表现出异源交叉反应。氨基酸分析表明两种酶之间没有主要的组成差异。在30℃时，正向和反向反应的类似Km和Ki值也基本相同。在30℃时，杜邦青霉酶的生理上重要的腺苷5'-磷酸硫酸（APS）合成活性为4 U mg蛋白质⁻¹，约为产黄青霉酶的一半。杜邦青霉酶在30℃时的钼解和ATP合成活性与产黄青霉酶相似。在50℃时，杜邦青霉酶的APS合成活性为12至19 U mg蛋白质⁻¹，高于产黄青霉酶在30℃时的活性（8±1 U mg蛋白质⁻¹）。在30℃非变性条件下用5,5'-二硫代双（2-硝基苯甲酸）（DTNB）处理产黄青霉酶，每个亚基修饰一个游离巯基。Vmax没有显著改变，但在低镁-ATP或SO₄²⁻（或MoO₄²⁻）存在下的催化活性明显降低。用四硝基甲烷进行化学修饰对动力学有相同的结果。天然的杜邦青霉酶在30℃和pH 8.0或pH 9.0时对DTNB或四硝基甲烷相对不反应，但在50℃和pH 8.0时，DTNB迅速修饰每个亚基的一个SH基团。产黄青霉的APS激酶（第二种硫酸激活酶）在42℃时解离成无活性的亚基。杜邦青霉的酶在42℃时保持完整且有活性。
