Hirata I Y, Boschcov P, Oliveira M C, Juliano M A, Miranda A, Chagas J R, Tsuboi S, Okada Y, Juliano L
Department of Biophysics, Escola Paulista de Medicina, São Paulo, Brazil.
Int J Pept Protein Res. 1991 Oct;38(4):298-307. doi: 10.1111/j.1399-3011.1991.tb01508.x.
The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.
为了研究假定的碳水化合物结合位点天冬酰胺-谷氨酸-丝氨酸（Asn-Glu-Ser）对人肾素水解作用的影响，合成了人血管紧张素原的N端十七肽（天冬氨酸-精氨酸-缬氨酸-酪氨酸-异亮氨酸-组氨酸-脯氨酸-苯丙氨酸-组氨酸-亮氨酸-缬氨酸-异亮氨酸-组氨酸-天冬酰胺-谷氨酸-丝氨酸-苏氨酸-NH2），其C端羧基被酰胺化。合成采用Honzl和Rudinger叠氮法通过片段缩合进行。在我们的叠氮片段缩合条件下，已证明在大多数缩合反应中组氨酸消旋化可忽略不计。人肾素在pH 7.3和37℃时从h-血管紧张素原（1-17）-NH2释放血管紧张素I，Km值为3.4×10⁻⁵M，这与不含碳水化合物结合位点的类似物h-血管紧张素原（1-13）相似。然而，4.1×10⁻⁹mol/酶活力单位·分钟的Vmax值高一个数量级。已证明猪胃蛋白酶优先切割亮氨酸¹⁰-缬氨酸¹¹键，并且令人惊讶的是，也切割组氨酸⁹-亮氨酸¹⁰键。
