KINETICS AND MECHANISM OF AUTOPROCESSING OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE FROM AN ANALOG OF THE GAG-POL POLYPROTEIN
KINETICS AND MECHANISM OF AUTOPROCESSING OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE FROM AN ANALOG OF THE GAG-POL POLYPROTEIN
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DOI：
10.1073/pnas.91.17.7970
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发表时间：
1994-08-16
影响因子：
11.1
通讯作者：
JERINA, DM
中科院分区：
文献类型：
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作者：
LOUIS, JM;NASHED, NT;JERINA, DM
复性后，MBP-Delta Tf-Protease-Delta Pol由HIV-1蛋白酶和转框蛋白(Delta Tf)的天然短序列和融合到大肠杆菌麦芽糖结合蛋白(MBP)的聚合酶(Delta Pol)组成，经过两步自动加工产生成熟的蛋白酶。最初的步骤对应于N-末端序列的切割，以释放蛋白质中间产物-Delta Pol，其酶活性与成熟酶相当。随后，成熟的酶由C末端较慢的裂解形成。酶活性增加的速度与MBP-Delta Tf出现和MBP-Delta Tf-Protease-Delta Pol消失的速度一致。初始速率与蛋白质浓度呈线性关系，表明N-末端的裂解是蛋白质浓度的一级反应。该反应被胃抑素A竞争性抑制，并且具有与成熟酶相似的pH速率曲线。这些结果和分子模拟研究从一个机制的角度进行了讨论，其中二聚体全长融合蛋白必须在N末端序列的限速分子内切割之前形成，从而导致酶活性的增加。
Upon renaturation, the polyprotein MBP-Delta TF-Protease-Delta Pol, consisting of HIV-1 protease and short native sequences from the trans-frame protein (Delta TF) and the polymerase (Delta Pol) fused to the maltose-binding protein (MBP) of Escherichia coli, undergoes autoprocessing to produce the mature protease in two steps. The initial step corresponds to cleavage of the N-terminal sequence to release the protein intermediate Protease-Delta Pol, which has enzymatic activity comparable to that of the mature enzyme. Subsequently, the mature enzyme is formed by a slower cleavage at the C terminus. The rate of increase in enzymatic activity is identical to that of the appearance of MBP-Delta TF and the disappearance of the MBP-Delta TF-Protease-Delta Pol. Initial rates are linearly dependent on the protein concentration, indicating that the N-terminal cleavage is first-order in protein concentration. The reaction is competitively inhibited by pepstatin A and has a pH rate profile similar to that of the mature enzyme. These results and molecular modeling studies are discussed in terms of a mechanism in which a dimeric full-length fusion protein must form prior to rate-limiting intramolecular cleavage of the N-terminal sequence that leads to an increase in enzymatic activity.