MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

逆转录酶（RT）和蛋白酶的作用机制 人类免疫缺陷病毒1型（HIV-1）正在进行研究， 开发这些酶的特异性抑制剂的目标。 （一） 来自构建体MBP-TF-PR-Pol的HIV-1蛋白酶的自动加工， 含有侧翼为19个氨基酸P01的蛋白酶序列（PR）， C-末端的12个氨基酸序列和反式 框架肽（TF）在N-末端，融合到麦芽糖结合 E.大肠杆菌，涉及两个连续的蛋白水解步骤： 在蛋白酶序列的N-末端裂解以释放13.2-kDa的 蛋白质中间体，然后C-末端裂解以得到成熟的， 11-kDa蛋白酶。 纯化的13.2-kDa中间体实际上是 在动力学行为上与成熟蛋白酶相同，但它 对尿素变性比成熟酶更敏感。 没有证据表明酶活性， 这种中间体的二聚体形式转化为蛋白质上的无活性单体 浓度高于25 nM。 自从自发转换的 成熟蛋白酶的中间产物是二级蛋白质 浓度，其机制可能涉及水解的C- 第二个二聚体将一个二聚体孕激素的末端片段连接到第二个二聚体。 ii）动力学 稳定状态的单核苷酸残基的掺入， 胸苷或4-硫代胸苷三磷酸（分别为TTP或4S-TTP） 将来自HIV-1的RNA-DNA模板-引物与那些 E.大肠杆菌DNA聚合酶I与DNA模板- 引物 对于Klenow片段，4S-TTP的米氏常数为： 3.5比TTP大10倍，而对于HIV-1 RT，Michaelis 两种三磷酸盐的澄清剂基本相同。 与 Klenow片段，该酶的周转速率常数为约。5 4S-TTP比其含氧类似物大一倍，而 对于RT，4S-TTP的该速率常数是观察到的TTP的速率常数的一半。 的 营业额的差异可能反映了 含氧和含硫寡核苷酸的解离 酶的产物，因为这一步很可能是速率- 确定酶促反应。

Mechanisms of action of the reverse transcriptase (RT) and protease of human immunodeficiency virus type 1 (HIV-1) are being investigated with the goal of developing specific inhibitors for these enzymes. i) Autoprocessing of the HIV-1 protease from a construct, MBP-TF-PR-Pol, containing the protease sequence (PR) flanked by a 19-amino acid P0l sequence at the C-terminus and a 12-amino acid sequence from the trans frame peptide (TF) at the N-terminus, fused to the maltose binding protein (MBP) of E. coli, involves two sequential proteolytic steps: cleavage at the N-terminus of the protease sequence to release a 13.2-kDa protein intermediate followed by C-terminal cleavage to give the mature, 11-kDa protease. The purified 13.2-kDa intermediate is virtually identical to the mature protease in its kinetic behavior, but it is somewhat more sensitive to denaturation by urea than the mature enzyme. There is no evidence for dissociation of the enzymatically active, dimeric form of this intermediate to inactive monomers at protein concentrations above 25 nM. Since the spontaneous conversion of the intermediate to the mature protease is second-order in protein concentration, its mechanism probably involves hydrolysis of the C- terminal fragment of one dimeric progein by a second dimer. ii) Kinetics of steady-state incorporation of a single nucleotide residue derived from thymidine or 4-thiothymidine triphosphate (TTP or 4S-TTP respectively) into a RNA-DNA template-primer by RT from HIV-1 were compared with those for the Klenow fragment of E. coli DNA polymerase I with a DNA template- primer. With the Klenow fragment, the Michaelis constant for 4S-TTP is 3.5 times larger than that for TTP, whereas with HIV-1 RT, the Michaelis constrants for the two triphosphates are essentially identical. With the Klenow fragment, the rate constant for turn-over of the enzyme is ca. 5 times larger with 4S-TTP than with its oxygen-containing analog, whereas with RT this rate constant for 4S-TTP is half that observd for TTP. The differences in turnover presumbly reflect differences in rates of dissociation of the oxygen- and sulfur-containing oligonucleotide products from the enzymes, since this step is likely to be rate- determining for the enzymatic reaction.