HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE .1. INITIAL VELOCITY STUDIES AND KINETIC CHARACTERIZATION OF REACTION INTERMEDIATES BY O-18 ISOTOPE EXCHANGE
HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE .1. INITIAL VELOCITY STUDIES AND KINETIC CHARACTERIZATION OF REACTION INTERMEDIATES BY O-18 ISOTOPE EXCHANGE
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DOI：
10.1021/bi00098a023
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发表时间：
1991-08-27
期刊：
影响因子：
2.9
通讯作者：
MEEK, TD
中科院分区：
文献类型：
--
作者：
HYLAND, LJ;TOMASZEK, TA;MEEK, TD
HIV-1蛋白酶的肽水解反应采用四种寡肽底物，Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, ac - ar - ala - ser - gln - asn - tyr - pro - val - val - nh2， Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2和ac - arg - lys - ile - leu - ph - leu - asp - gly - nh2，它们类似于天然存在的多蛋白底物Pr55gag和Prl60gag-pol中的两个裂解位点。在pH 6.0、0.2 M NaCl和37℃条件下，动力学参数V/KE(t)和V/E(t)分别为0.16 ~ 7.5 mM-1 s-1和0.24 ~ 29 s-1。通过使用多种无机盐，发现离子强度的增加对肽解反应具有非特异性激活作用，V/K随离子强度的增加呈明显的抛物线型增加，而V则有轻微的增加或减少。从产物抑制研究来看，蛋白酶的动力学机制取决于所研究的底物，要么是随机的，要么是有序的。对于大多数寡肽底物，逆反应或部分逆反应（通过羧基产物转化为底物的同位素交换测量）可以忽略不计，但该酶催化Ac-Ser-Gln-Asn-Tyr和ph - leu - asp - gly - nh2生成Ac-Ser-Gln-Asn-Tyr- ph - leu - asp - gly - nh2。蛋白酶催化的O-18原子从(H2O)-O-18交换到重组底物的速率是正向肽水解反应的0.01-0.12倍。这些研究的结果与形成具有动力学能力的酶结合酰胺水合物中间体一致，其崩溃是反应途径中限速的化学步骤。
The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Prl60gag-pol. The values for the kinetic parameters V/KE(t) and V/E(t) were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37-degrees-C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength, V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of O-18 from (H2O)-O-18 into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.