MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

反转录酶和蛋白水解酶的作用机制 人类免疫缺陷病毒1型(HIV-1)正在接受研究 开发针对这些酶的特定抑制剂的目标。i) 从构建的MBP-Tf-PR-POL中自动加工HIV-1蛋白酶， 含有由19个氨基酸组成的P0侧翼的蛋白酶序列(PR C末端的序列和来自反式的12个氨基酸序列 N-末端的框架肽(Tf)，与麦芽糖结合融合 大肠杆菌的蛋白质(MBP)包括两个顺序的蛋白分解步骤： 蛋白水解酶序列N-末端的切割释放13.2-kDa 蛋白质中间体，然后C-端裂解，得到成熟的， 11-kDa蛋白水解酶。纯化的13.2 kDa中间体几乎是 与成熟的蛋白酶在动力学行为上是相同的，但它是 对尿素的变性比成熟的酶更敏感。 没有证据表明酶活性的解离， 蛋白质中这种中间到非活性单体的二聚体形式 浓度在25纳米以上。因为他们的自发转换 在成熟的蛋白酶中间是蛋白质的二级结构。 浓度，其机制可能涉及C-水解酶- 一个二聚体的末端片段由第二个二聚体组成。Ii)动力学 稳定掺入来源于以下来源的单核苷酸残基 胸腺嘧啶核苷或4-硫代胸苷三磷酸(分别为TTP或4S-TTP) 用RT方法从HIV-1中扩增成RNA-DNA模板-引物，并与 对于带有DNA模板的大肠杆菌DNA聚合酶I的Klenow片段- 底漆。对于Klenow片段，4S-TTP的米氏常数为 比TTP大3.5倍，而使用HIV-1 RT，米氏 这两种三磷酸盐的构造物基本相同。与 Klenow片段，酶的翻转速率常数约为5。 使用4S-TTP比使用其含氧类似物大几倍，而 在RT下，4S-TTP的这个速率常数是TTP观察到的速率常数的一半。这个 营业额的差异可能反映了 含氧和含硫寡核苷酸的解离 来自酶的产物，因为这一步很可能被分级- 用于酶反应的测定。

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.