TRANSITION OF BOVINE TRYPSINOGEN TO A TRYPSIN-LIKE STATE UPON STRONG LIGAND-BINDING .2. BINDING OF THE PANCREATIC TRYPSIN-INHIBITOR AND OF ISOLEUCINE-VALINE AND OF SEQUENTIALLY RELATED PEPTIDES TO TRYPSINOGEN AND TO P-GUANIDINOBENZOATE-TRYPSINOGEN
TRANSITION OF BOVINE TRYPSINOGEN TO A TRYPSIN-LIKE STATE UPON STRONG LIGAND-BINDING .2. BINDING OF THE PANCREATIC TRYPSIN-INHIBITOR AND OF ISOLEUCINE-VALINE AND OF SEQUENTIALLY RELATED PEPTIDES TO TRYPSINOGEN AND TO P-GUANIDINOBENZOATE-TRYPSINOGEN
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DOI：
10.1016/0022-2836(79)90227-4
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发表时间：
1979-01-01
影响因子：
5.6
通讯作者：
BODE, W
中科院分区：
文献类型：
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作者：
BODE, W
PgB-胰酶原与Ile-Val结合后，其圆二色谱发生了特殊的变化，证明了它的构象发生了转变。这些变化被用来确定在不同的钙离子浓度下，顺序类似于牛胰酶N-端或几种胰酶原的N-端激活肽序列的各种多肽的结合常数。在所有研究的多肽中，Ile-Val和Ile-Val-Gly表现出最强的结合亲和力。用其他氨基酸取代第一个异亮氨酸或第二个缬氨酸残基，大大降低了多肽的亲和力。讨论是基于已知的Ile16-Vall7-Gly18 N端和Ile16裂隙中Ile-Val二肽的空间排列(牛胰蛋白酶和胰蛋白酶原-PTI(胰腺胰蛋白酶抑制物)-Ile-Val复合体的晶体结构；Bode等人，1978)。第一个和第二个肽残基的结合自由能几乎是相加的，表明亚基之间是独立的。第三个残基甘氨酸对结合没有显著贡献。各种胰酶原N末端的多肽类似物对Ile16裂解没有可测量的亲和力。在钙离子存在和不存在的情况下，利用PTI与α-糜蛋白酶的竞争结合，测定了PTI与胰蛋白酶原结合的平衡常数和Ile-Val对所生成的二元络合物的亲和力。这些竞争实验允许估计由于胰酶N末端和多肽类似物结合到预先形成的Ile16裂隙以及由于共价连接的PGB基团特异性地埋入固定的特异性口袋而导致的胰酶原构象转变到类胰酶状态所引起的标准自由能变化。根据-43kJ·mol~(-1)的自由能变化耦合，这个口袋与Ile16裂隙协同连接。
p-Guanidinobenzoate(pGB)-trypsinogen is transformed into a tryspin-like conformation upon binding of Ile-Val as evidenced by specific changes in its circular dichroism spectrum. The changes were used to determine the association constants for the binding of a variety of peptides sequentially analogous to either the bovine trypsin N-terminus or to the N-terminal activation peptide sequences of several trypsinogens at different Ca2+ concentrations. Ile-Val and Ile-Val-Gly exhibit the strongest binding affinity of all peptides investigated. Replacement of the 1st isoleucine or of the 2nd valine residue by other amino acids considerably reduces the peptide affinity. Discussion is based on the known spatial arrangement of the Ile16-Vall7-Gly18 N-terminus and of the Ile-Val dipeptide in the Ile16 cleft (crystal structures of bovine trypsin and of the trypsinogen-PTI (pancreatic trypsin inhibitor)-Ile-Val complex; Bode et al., 1978). The free energies of binding of the 1st and of the 2nd peptide residue are almost additive indicating independency between the subsites. The 3rd residue, glycine, does not significantly contribute to binding. The peptide analogs of various trypsinogen N-termini exhibit no measurable affinity for the Ile16 cleft. The equilibrium constant for the binding of PTI to trypsinogen and the affinity of Ile-Val for the resulting binary complex were determined in the presence and absence of Ca2+, using the competitive PTI-binding to .alpha.-chymotrypsin. These competition experiments allow the estimation of the standard free-energy changes due to the conformational transition of trypsinogen into a trypsin-like state due to the binding of the trypsin N-terminus and of the peptide analogs into the preformed Ile16 cleft, and due to the specific burying of the covalently linked pGB group in the fixed specificity pocket. This pocket is cooperatively linked with the Ile16 cleft according to a free-energy change coupling of -43 kJ mol-1.