Ingram S W, Stratemann S A, Barnes L D
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760, USA.
Biochemistry. 1999 Mar 23;38(12):3649-55. doi: 10.1021/bi982951j.
The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.
裂殖酵母粟酒裂殖酵母在第一条染色体上含有一个基因，该基因编码一种假定的Nudix水解酶YA9E。这个名为aps1的基因已被克隆，其蛋白质已从大肠杆菌中纯化出来，每升培养物的Aps1产量为10毫克。Aps1由210个氨基酸组成，计算分子量为23724道尔顿，通过分析超速离心法测定，其表现为单体，沉降系数为1.92 S。通过分析超速离心法和凝胶过滤色谱法测定，其有效流体动力学半径约为29埃。通过蛋白质免疫印迹法在粟酒裂殖酵母中检测到Aps1的表达，它是一种催化二核苷寡磷酸水解的酶，其中Ap6A和Ap5A是优选底物。主要反应产物是Ap6A产生的ADP和p4A以及Ap5A产生的ADP和ATP。Ap6A和Ap5A的Km值分别为19微摩尔和22微摩尔，相应的kcat值分别为2.0秒-1和1.7秒-1。该酶对Ap4A的活性有限，对Ap3A、ADP-核糖和NADH的活性可忽略不计。Aps1催化单核苷酸的水解，活性从p5A到AMP依次降低。以Ap6A为底物时，在pH 7.6和存在0.1-1 mM MnCl2的条件下观察到最佳活性。Aps1是从粟酒裂殖酵母中分离出的首个Nudix水解酶，也是首个被鉴定具有这种特定底物特异性和反应产物的酶。
