Skeggs L T, Lentz K E, Kahn J R, Hochstrasser H
J Exp Med. 1968 Jul 1;128(1):13-34. doi: 10.1084/jem.128.1.13.
A number of peptides have been synthesized which represent portions of the tetradecapeptide renin substrate molecule, and which contain the hydrolyzable leu-leu bond. An automatic chemical method for determination of the velocity of the reaction of renin with these compounds was developed. Application of the method at several levels of substrate concentration permitted construction of Lineweaver-Burk plots, and calculation of Michaelis constants (K(m)) and maximal velocities (V(max)). The results show that the maximum affinity of the enzyme (lowest K(m)) for substrate is achieved only with the full tetradecapeptide molecule (asp(1)-arg(2)-val(3)-tyr(4)-ileu(5)-his(6)-pro(7)-phe(8)-his(9)-leu(10)-leu(11)-val(12)-tyr(13)-ser(14)). Removal of asp(1) and arg(2) from the N-terminal increases the K(m) eight-fold. Further, moderate increase in K(m) occurs when the next amino acids, val(3), tyr(4) and ileu(5), are removed. The further removal of his(6) results in a marked reduction in the V(max). Removal of ser(14) from the C-terminal of the nonapeptide his(6)-pro(7)-phe(8)-his(9)-leu(10)-leu(11)-val(12)-tyr(13)-ser(14) does not greatly affect the K(m) nor the V(max). Further removal of tyr(13) from this compound results in complete loss of substrate activity. It is suggested that the compounds his(6)-pro(7)-phe(8)-his(9)-leu(10)-leu(11)-val(12)-tyr(13)-ser(14) or his(6)-pro(7)-phe(8)-his(9)-leu(10)-leu(11)-val(12)-tyr(13) might be used as substrates for the chemical assay and standardization of renin.
已合成了多种肽，它们代表十四肽肾素底物分子的部分片段，并且含有可水解的亮氨酸 - 亮氨酸键。开发了一种自动化学方法来测定肾素与这些化合物反应的速度。在几个底物浓度水平上应用该方法可以构建林-贝氏图，并计算米氏常数（K(m)）和最大速度（V(max)）。结果表明，只有完整的十四肽分子（天冬氨酸(1)-精氨酸(2)-缬氨酸(3)-酪氨酸(4)-异亮氨酸(5)-组氨酸(6)-脯氨酸(7)-苯丙氨酸(8)-组氨酸(9)-亮氨酸(10)-亮氨酸(11)-缬氨酸(12)-酪氨酸(13)-丝氨酸(14)）才能使酶对底物具有最大亲和力（最低的K(m)）。从N端去除天冬氨酸(1)和精氨酸(2)会使K(m)增加8倍。此外，当去除接下来的氨基酸缬氨酸(3)、酪氨酸(4)和异亮氨酸(5)时，K(m)会适度增加。进一步去除组氨酸(6)会导致V(max)显著降低。从九肽组氨酸(6)-脯氨酸(7)-苯丙氨酸(8)-组氨酸(9)-亮氨酸(10)-亮氨酸(11)-缬氨酸(12)-酪氨酸(13)-丝氨酸(14)的C端去除丝氨酸(14)对K(m)和V(max)影响不大。从该化合物中进一步去除酪氨酸(13)会导致底物活性完全丧失。有人提出，化合物组氨酸(6)-脯氨酸(7)-苯丙氨酸(8)-组氨酸(9)-亮氨酸(10)-亮氨酸(11)-缬氨酸(12)-酪氨酸(13)-丝氨酸(14)或组氨酸(6)-脯氨酸(7)-苯丙氨酸(8)-组氨酸(9)-亮氨酸(10)-亮氨酸(11)-缬氨酸(12)-酪氨酸(13)可作为肾素化学测定和标准化的底物。
