Elimination of the sensitivity of L-aspartase to active-site-directed inactivation without alteration of catalytic activity.
Elimination of the sensitivity of L-aspartase to active-site-directed inactivation without alteration of catalytic activity.
复制标题
消除 L-天冬氨酸酶对活性位点定向失活的敏感性，而不改变催化活性。
DOI：
10.1021/bi00011a006
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Viola,RE
中科院分区：
文献类型：
--
作者：
Giorgianni,F;Beranová,S;Wesdemiotis,C;Viola,RE
1994年12月16日收到的修订版Mandarin pt ®摘要：大肠杆菌中L-丝氨酸蛋白酶的催化活性先前已被证明对巯基试剂敏感。然而，使用组特异性试剂，以及在半胱氨酸蛋白酶-半胱氨酸蛋白酶家族的酶之间的序列同源性比较研究，还没有导致鉴定出特定的、必需的半胱氨酸残基。我们最近已经表明，L-天冬氨酸-L-天冬氨酸半醛是L-精氨酸酶的另一种底物，产生特异性灭活该酶的富马酸半醛（FAA）[Schindler，J.F.，和Viola，RE（1994）Biochemistry 33，9365]。所得到的失活酶的蛋白水解酶谱现已通过HPLC和质谱法绘制。一个特定的残基（Cys-273）已被确定为FAA修饰的网站。在E.大肠杆菌酶已经产生了改变的酶，其对活性位点定向失活的敏感性大大降低，同时保留了完全的催化活性。因此，半胱氨酸-273已被鉴定为活性位点亲核试剂，其虽然不直接参与L-天冬氨酸酶中的催化，但准备攻击酶结合产物类似物中的活化双键。1.1)催化L-天冬氨酸的可逆脱氨作用，形成富马酸和氨。来自大肠杆菌的酶是四聚体的，具有aspA基因的序列（Takagi等人，1985）编码由477个氨基酸组成的亚基，分子量为52224。
Revised Manuscript Received December 16, 1994® abstract: The catalytic activity of the enzyme L-aspartase from Escherichia coli has previously been shown to be sensitive to sulfhydryl reagents. The use of group-specific reagents, and a sequence homology comparison study among the fumarase—aspartase family of enzymes, has not, however, lead to the identification of a specific, essential cysteinyl residue. We have recently shown that L-aspartate-/?-semialdehyde is an alternative substrate for L-aspartase, producing fumaric acid semialdehyde (FAA) which specifically inactivates the enzyme [Schindler, J. F., & Viola, RE (1994) Biochemistry 33, 9365]. Proteolytic digests of the resulting inactivated enzyme have now been mapped by HPLC and mass spectrometry. A specific residue (Cys-273) has been determined to be the site of FAA modification. Site-directed mutagenesis of this cysteine in the E. coli enzyme has produced altered enzymes which are considerably less sensitive to active-site-directed inactivation, while retaining full catalytic activity. Thus, cysteine-273 has been identified as an active-site nucleophile that, while not directly involved in catalysis in L-aspartase, is poised to attack an activated double bond in an enzyme-bound product analogue.L-Aspartase (L-aspartate ammonia-lyase, EC 4.3. 1.1) cata-lyzes the reversible deamination of L-aspartic acid to form fumaric acid and ammonia. The enzyme from Escherichia coli is tetrameric, with the sequence of the aspA gene (Takagi et al., 1985) encoding a subunit composed of 477 amino acids with a molecular weight of 52 224.