Yang J J, Artis D R, Van Wart H E
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304.
Biochemistry. 1994 May 31;33(21):6516-23. doi: 10.1021/bi00187a019.
The effect of sodium halide salts on the hydrolysis of three of the dansyl (Dns) peptide substrates described in the previous paper (Yang & Van Wart, 1994) by thermolysin have been studied. Increasing concentrations of sodium chloride decrease the KM value for the hydrolysis of the tripeptides Dns-Gly-Phe-Ala and Dns-Ala-Phe-Ala but leave kcat unaltered. This kinetic behavior is described by a nonessential activation mechanism in which chloride binds preferentially to the enzyme-substrate complex. Similar trends are found for the sodium bromide and fluoride salts. In contrast, sodium chloride decreases both KM and kcat almost equally for the hydrolysis of Dns-Ala-Ala-Phe-Ala, leaving kcat/KM unchanged. Thus, chloride is an uncompetitive inhibitor of this substrate. Molecular modeling studies have been carried out in order to explain the effect of chloride on the binding of these dansyl peptides. The decrease in KM for the hydrolysis of all three substrates is attributed to an interaction of chloride with Arg-203 located in the active site to stabilize the enzyme-substrate complexes. The differential effect of chloride on the kcat values for the hydrolysis of the dansyl tripeptides vs dansyl tetrapeptide is related to differences in binding on the Pn side of the substrates. The tripeptides are predicted to bind to the active site of thermolysin in a single low-energy conformation. However, there are two populations of low-energy binding modes for the tetrapeptide, one of which is believed to be a more productive binding mode.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了卤化钠盐对前一篇论文（Yang & Van Wart，1994）中描述的三种丹磺酰（Dns）肽底物被嗜热菌蛋白酶水解的影响。氯化钠浓度的增加降低了三肽Dns-Gly-Phe-Ala和Dns-Ala-Phe-Ala水解的KM值，但kcat不变。这种动力学行为由一种非必需激活机制描述，其中氯离子优先结合到酶-底物复合物上。溴化钠和氟化钠盐也有类似趋势。相比之下，氯化钠对Dns-Ala-Ala-Phe-Ala水解的KM和kcat几乎有同等程度的降低，kcat/KM不变。因此，氯离子是该底物的非竞争性抑制剂。已进行分子模拟研究以解释氯离子对这些丹磺酰肽结合的影响。所有三种底物水解的KM降低归因于氯离子与位于活性位点的Arg-203相互作用以稳定酶-底物复合物。氯离子对丹磺酰三肽与丹磺酰四肽水解kcat值的不同影响与底物Pn侧结合的差异有关。预测三肽以单一低能构象结合到嗜热菌蛋白酶的活性位点。然而，四肽有两种低能结合模式群体，其中一种被认为是更有效的结合模式。（摘要截断于250字）
