Ariyananda Lushanti De Zoysa, Lee Peychii, Antonopoulos Christina, Colman Roberta F
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2009 Jun 16;48(23):5291-302. doi: 10.1021/bi802321m.
Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The V(max) (at 25 degrees C) for R194C is comparable to that of WT, while those of L311V, R396C, R396H, and K246E are considerably reduced and affinity for adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity, whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 degrees C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60 degrees C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 degrees C and after incubation at 37 degrees C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme's structure and/or native conformation which are required for catalytic function.
腺苷酸琥珀酸裂解酶（ASL）是嘌呤生物合成关键反应的催化剂，通常是一种同四聚体，其中三个亚基构成四个活性位点中的每一个。人类ASL缺乏症是一种与自闭症和智力迟钝相关的遗传性代谢疾病。我们已经对五个与疾病相关的ASL突变体进行了表征：R194C和K246E位于亚基界面，L311V位于远离活性位点的中央螺旋区域，R396C和R396H位于活性位点入口处。R194C在25℃时的V(max)与野生型相当，而L311V、R396C、R396H和K246E的V(max)则显著降低，且对腺苷酸琥珀酸的亲和力得以保留。与野生型相比，突变酶的正协同性降低。K246E主要以二聚体或单体形式存在，这解释了其可忽略不计的活性，而其他突变酶在四聚体占主导方面与野生型相似。在37℃时，野生型和这些突变酶的比活性随时间缓慢降低30 - 40%，并达到极限比活性，而四聚体的量没有显著变化。突变体R194C的独特之处在于，在60℃这个更严苛的温度下会迅速失活，这表明它是体外最不稳定的酶。从25℃以及在37℃孵育后的蛋白质荧光强度可以明显看出突变酶的构象变化，这与酶活性的丧失相关。因此，这些与疾病相关的单突变可通过影响活性位点或扰乱酶的结构和/或催化功能所需的天然构象来产生活性降低的酶。
