Why does threonine, and not serine, function as the active site nucleophile in proteasomes?
Why does threonine, and not serine, function as the active site nucleophile in proteasomes?
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DOI：
10.1074/jbc.275.20.14831
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发表时间：
2000-05-19
影响因子：
4.8
通讯作者：
Goldberg, AL
中科院分区：
文献类型：
--
作者：
Kisselev, AF;Songyang, Z;Goldberg, AL
蛋白酶体属于酰胺酶的N-末端亲核基团，通过一种新的蛋白水解机制发挥作用，其中N-末端苏氨酸的羟基是催化亲核基团。然而，尚不清楚为什么苏氨酸在所有蛋白酶体活性位点中是保守的，因为其在来自嗜酸热浆菌（T1 S突变体）的蛋白酶体中被丝氨酸取代不会改变Suc-LLVY-amc的水解速率（Seemuller，E.，Lupas，A.，Stock，D.，J.洛，胡贝尔河，Baumeister，W.（1995）Science 268，579-582）和其他标准肽酰胺底物。然而，我们发现十肽文库中的真正肽键被T1 S突变体切割比野生型（wt）蛋白酶体慢10倍。在降解蛋白质时，T1 S蛋白酶体比wt慢3.5- 6倍。当用蛋白酶体激活核苷酸酶（PAN）ATP酶复合物刺激蛋白水解时，这种差异增加。与野生型蛋白酶体不同，突变型蛋白酶体的蛋白质分解中，肽键断裂似乎是限速的。令人惊讶的是，肽酯被两种颗粒水解得比相应的酰胺快得多，并且T1 S突变体比wt.此外，T1 S突变体失活的酯抑制剂断裂-lactacystin-β-内酯几倍快于野生型，但与非酯不可逆抑制剂以相似的速率反应。T1 A和T1 C突变体在所有这些测定中完全失活。因此，蛋白酶体缺乏额外的活性位点，N-末端苏氨酸进化，因为它比丝氨酸更有效地分解蛋白质。
Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemuller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates, However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes, In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.