Thomsen N K, Christensen U, Madsen K, Sottrup-Jensen L
Department of Molecular Biology, University of Arhus, Denmark.
Eur J Biochem. 1992 Oct 1;209(1):409-14. doi: 10.1111/j.1432-1033.1992.tb17303.x.
The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.
通过高效液相色谱法（HPLC）测量裂解产物随时间的生成情况，研究了胰蛋白酶与人α2-巨球蛋白（α2M）诱饵区（666-706位氨基酸残基）中一段序列（676-703位氨基酸残基）对应的合成肽之间相互作用的时间进程。该合成肽中有三个胰蛋白酶的主要裂解位点。最快的裂解发生在α2M中对应于Arg696-Leu的肽键处，估计的kcat/Km = 1 - 2×10⁶ M⁻¹·s⁻¹。考虑到α2M是四聚体，该值与表征α2M和胰蛋白酶相互作用的值大小相同，kcat/Km = 5×10⁶ M⁻¹·s⁻¹ [克里斯蒂安森，U. & 索特鲁普-詹森，L.（1984年）《生物化学》23，6619 - 6626]。α2M中对应于Arg681-Val和Arg692-Gly的肽键处裂解的kcat/Km值分别为1.5×10⁵ M⁻¹·s⁻¹和1.3×10⁵ M⁻¹·s⁻¹。中间产物肽的裂解较慢，kcat/Km在13 - 1.3×10⁶ M⁻¹·s⁻¹范围内。合成肽中快速裂解的Km值为8 - 10 μM。¹H-NMR光谱表明该肽没有有序结构。因此，该肽的非常快速的裂解与一种松散结构相符，这种结构很容易采用有利于胰蛋白酶识别和裂解的构象。
