Wu J C, Carr S F, Jarnagin K, Kirsher S, Barnett J, Chow J, Chan H W, Chen M S, Medzihradszky D, Yamashiro D
Syntex Research, Palo Alto, California 94304.
Arch Biochem Biophys. 1990 Mar;277(2):306-11. doi: 10.1016/0003-9861(90)90584-l.
A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.
采用固相法合成了一种由99个氨基酸组成的蛋白质，其推导序列来自2型人类免疫缺陷病毒（HIV - 2）的蛋白酶，并对其特异性进行了检测。折叠后的肽催化HIV - 1重组43 kDa GAG前体蛋白（F - 16）的特异性加工。尽管HIV - 2蛋白酶与HIV - 1蛋白酶的氨基酸同一性仅为48%，但HIV - 2酶对HIV - 1 GAG前体表现出相同的特异性。与蛋白酶孵育的F - 16 GAG产生了34、32、24、10和9 kDa的片段。蛋白水解片段的N端氨基酸序列分析表明，HIV - 2蛋白酶识别的切割位点与HIV - 1蛋白酶的相同。F - 16 GAG中经证实的切割位点似乎是独立加工的，这从几乎等量的中间片段P32和P34的形成可以看出。两个病毒GAG蛋白之间最靠近氨基末端的位点相当保守（HIV - 1中为...VSQNY - PIVQN...，HIV - 2中为...KGGNY - PVQHV...）。相比之下，HIV - 2 GAG假定的第二个位点（...IPFAA - AQQKG...）与HIV - 1 GAG的位点2（...SATIM - MQRGN...）的序列同一性最小。底物中的这些序列变异表明可能影响蛋白酶识别的高阶结构特征。胃蛋白酶抑制剂A抑制HIV - 2蛋白酶，而1,10 - 菲咯啉和苯甲基磺酰氟则不抑制；这些结果与HIV和其他逆转录病毒的蛋白酶是天冬氨酸蛋白酶这一发现一致。
