Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase
Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase
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DOI：
10.1021/bi101832w
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发表时间：
2011-04-12
期刊：
影响因子：
2.9
通讯作者：
Georgiou, George
中科院分区：
文献类型：
--
作者：
Cantor, Jason R.;Stone, Everett M.;Georgiou, George
N-末端天冬酰胺水解酶（NTAN 1）对N-末端L-Asn的酶促脱酰胺作用是蛋白质降解的泛素依赖性N-末端规则途径的特征，其将蛋白质的体内半衰期与其N-末端残基的身份相关联。在此，我们报告的细菌表达，纯化，和生化特性的人NTAN 1（hNTAN 1）。我们在这里表明，hNTAN 1是高度选择性的水解N-末端肽基L-Asn，但不能脱酰胺游离L-Asn或L-Gln，N-末端肽基L-Gln，或乙酰化的N-末端肽基L-Asn。类似于其他N-末端脱酰胺酶，hNTAN 1被证明具有一个关键的Cys残基，这是催化所绝对需要的，部分证实了通过Cys 75 Ala点突变废除活性。我们还提出的证据表明，一个保守的L-脯氨酸在N-末端的hNTAN 1的起始，L-蛋氨酸去除后的曝光是重要的酶的功能。这里提出的结果应有助于阐明在其他研究中观察到的NTAN 1缺陷小鼠的神经缺陷的分子机制，并在通过N-末端规则途径调节蛋白质周转的酶靶向的潜在生理底物的发现。
The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating, L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway.