Li M, Luo W, White E H
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Arch Biochem Biophys. 1995 Jun 20;320(1):135-40. doi: 10.1006/abbi.1995.1351.
alpha-Chymotrypsin was irreversibly inhibited with an enzyme-activated N-nitrosamide inhibitor, N-nitroso-N-(1-naphthylmethyl)-N'-isobutyrylalanine; alkylation of the active-site residues by the naphthylmethyl cation produced in the enzymatic reaction occurred. The inhibited enzyme was reduced and aminoethylated and then subjected to tryptic and chymotryptic digestion. Separation of the digest by reversed-phase HPLC revealed one major new peak relative to that of a control run from the native enzyme. Subsequent amino acid analysis and sequencing along with fast atom bombardment-mass spectrometry measurements indicated that this new peak stemmed from an active-site peptide, Met-192-Leu-199, and that the naphthylmethyl label was attached to the side-chain oxygen of Ser-195. The general approach employed can be applied to labeling active sites of a variety of hydrolytic enzymes.
α-胰凝乳蛋白酶被一种酶激活的N-亚硝基酰胺抑制剂N-亚硝基-N-(1-萘甲基)-N'-异丁酰丙氨酸不可逆地抑制；在酶促反应中产生的萘甲基阳离子使活性位点残基发生烷基化。将受抑制的酶进行还原和氨乙基化，然后用胰蛋白酶和胰凝乳蛋白酶进行消化。通过反相高效液相色谱法分离消化产物，与天然酶的对照运行相比，显示出一个主要的新峰。随后的氨基酸分析和测序以及快原子轰击质谱测量表明，这个新峰来自一个活性位点肽段，即Met-192-Leu-199，并且萘甲基标记连接在Ser-195的侧链氧上。所采用的一般方法可应用于标记各种水解酶的活性位点。
