Substrates and inhibitors of human T-cell leukemia virus type I protease
Substrates and inhibitors of human T-cell leukemia virus type I protease
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DOI：
10.1021/bi982004a
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发表时间：
1998-12-15
期刊：
影响因子：
2.9
通讯作者：
Ikeda, RA
中科院分区：
文献类型：
--
作者：
Ding, YS;Rich, DH;Ikeda, RA
HTLV-I是一种与成人T细胞白血病相关的致癌逆转录病毒。HTLV-I酶和HTLV-I酶与含有先导肽的十碳组氨酸融合蛋白(His-Protein)已被克隆、表达和纯化。复性后的蛋白水解酶具有较高的活性，表现出几乎相同的活性。为了开始表征HTLV-I的特异性，我们测量了多肽底物的蛋白酶裂解和蛋白酶抑制剂的抑制作用。HTLV-I酶切代表HTLV-I逆转录病毒加工位点P19/24的多肽(APQVLPVMHPHG)的K-m和k(CAT)值分别为470mM和0.184 S(-1)，而代表加工位点P24/15的多肽(KTKVLVVQPK)的K-m和k(CAT)值分别为310mM和0.0060 S(-1)。用对硝基苯丙氨酸(NPH)取代P19/24中的P1‘Pro，可提高HTLV-I酶对底物(APQVLNphVMHPL)的切割能力。同时考察了一系列蛋白酶抑制剂对HTLV-I和His-蛋白水解酶的抑制作用。结果表明，一系列胃酶抑制剂抑制HTLV-I和His-蛋白酶的K-I值在7~10 mU M之间，而HIV-1酶抑制剂的K-I值在6~127 mU M之间。抑制HIV-1蛋白酶的Ki值为0.24~1.0mM。提示HTLV-I酶的底物结合部位与胃酶和HIV-1酶的底物结合部位不同，目前使用的HIV-1酶抑制剂对HTLV-I感染的治疗效果不佳。
HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded K-m and k(cat) values of 470 mu M and 0.184 s(-1) while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded K-m and k(cat) values of 310 mu M and 0.0060 s(-1). When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the K-i values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 mu M, while the K-i values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 mu M. In comparison, the K-i values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 mu M. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.