Schock H B, Garsky V M, Kuo L C
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
J Biol Chem. 1996 Dec 13;271(50):31957-63. doi: 10.1074/jbc.271.50.31957.
Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W. A., Blahy, O. M. , Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). These four substitutions are among the prominent mutations found in primary HIV isolates obtained from patients undergoing therapy with several protease inhibitors. Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110-360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing Ki), and those away from the active site enhance catalysis (by increasing kcat/KM). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively dictated by those of the double mutants.
人类免疫缺陷病毒1型（HIV-1）蛋白酶中仅四个氨基酸（M46I/L63P/V82T/I84V）的位点特异性替换就会导致对一组蛋白酶抑制剂产生交叉耐药性，这些抑制剂要么处于临床试验阶段，要么最近已被批准用于HIV治疗（康德拉，J. H.，施莱夫，W. A.，布拉希，O. M.，加德雷尔斯基，L. J.，格雷厄姆，D. J.，金特罗，J. C.，罗兹，A.，罗宾斯，H. L.，罗斯，E.，希瓦普拉卡什，M.，提图斯，D.，杨，T.，特普勒，H.，斯奎尔斯，K. E.，多伊奇，P. J.，以及埃米尼，E. A.（1995年）《自然》374卷，569 - 571页）。这四个替换是在从接受几种蛋白酶抑制剂治疗的患者中分离出的原发性HIV毒株中发现的主要突变。其中两个突变（V82T/I84V）位于酶的结合裂隙内，而另外两个（M46I/L63P）则远离该裂隙。现在已经根据这些突变对蛋白酶结合亲和力和催化效率造成的影响来描述它们的功能作用。我们发现，M46I和L63P的双重替换不影响结合，但反而赋予该酶显著超过野生型酶（110 - 360%）的催化效率。相比之下，V82T和I84V的双重替换对蛋白酶的结合能力以及催化能力都有损害。当这四个氨基酸替换组合在一起时，会使蛋白酶产生对抑制剂耐药，并且具有比仅在其活性位点含有突变的蛋白酶更高的催化活性。结果表明，在产生耐药性方面，蛋白酶的这四个位点特异性突变在功能上具有补偿作用；活性位点的突变降低平衡结合（通过增加Ki），而远离活性位点的突变增强催化作用（通过增加kcat/KM）。这一结论在能量估算方面得到了进一步支持，因为四重突变体的结合和催化吉布斯自由能在数量上由双重突变体的吉布斯自由能决定。
