Ding Y S, Rich D H, Ikeda R A
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.
Biochemistry. 1998 Dec 15;37(50):17514-8. doi: 10.1021/bi982004a.
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 Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM 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 Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki 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 microM. 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.
人类嗜T淋巴细胞病毒I型（HTLV-I）是一种致癌逆转录病毒，与成人T细胞白血病相关。HTLV-I蛋白酶以及与含十组氨酸前导肽融合的HTLV-I蛋白酶（His-蛋白酶）已被克隆、表达和纯化。重折叠后的蛋白酶具有活性，且表现出几乎相同的酶活性。为了开始对HTLV-I的特异性进行表征，我们测定了肽底物的蛋白酶切割以及蛋白酶抑制剂的抑制作用。HTLV-I蛋白酶对代表HTLV-I逆转录病毒加工位点P19/24（APQVLPVMHPHG）的肽进行切割，得到的米氏常数（Km）和催化常数（kcat）分别为470微摩尔和0.184秒-1，而对代表加工位点P24/15（KTKVLVVQPK）的肽进行切割，得到的Km和kcat值分别为310微摩尔和0.0060秒-1。当P19/24的P1'脯氨酸被对硝基苯丙氨酸（Nph）取代时，HTLV-I蛋白酶切割底物（APQVLNphVMHPL）的能力得到了提高。还测试了一系列蛋白酶抑制剂对HTLV-I蛋白酶和His-蛋白酶的抑制作用。结果发现，一系列胃蛋白酶抑制剂对HTLV-I蛋白酶和His-蛋白酶的抑制常数（Ki）值在7纳摩尔至10微摩尔之间，而一系列HIV-1蛋白酶抑制剂的Ki值在6纳摩尔至127微摩尔之间。相比之下，胃蛋白酶抑制剂对胃蛋白酶的抑制Ki值在0.72至19.2纳摩尔之间，HIV蛋白酶抑制剂对HIV-1蛋白酶的抑制Ki值在0.24纳摩尔至1.0微摩尔之间。这些数据表明，HTLV-I蛋白酶的底物结合位点与胃蛋白酶和HIV-1蛋白酶的底物结合位点不同，并且目前使用的HIV-1蛋白酶抑制剂对治疗HTLV-I感染无效。
