Juliano M A, Filira F, Gobbo M, Rocchi R, Del Nery E, Juliano L
Department of Biophysics, Escola Paulista de Medicina, São Paulo, Brazil.
J Pept Res. 1999 Feb;53(2):109-19. doi: 10.1034/j.1399-3011.1999.00012.x.
We synthesized short chromogenic peptidyl-Arg-p-nitroanilides containing either (Galbeta)Ser or (Glcalpha,beta)Tyr at P2 or P3 sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl-X-Arg-pNa and Acetyl-X-Phe-Arg-pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe8-His-Leu-Val-Ile-His-Asn14 of human angiotensinogen, in which [GlcNAcbeta]Asn was introduced before Phe8 and/or after His13 and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]-ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P3 it is unfavorable, in contrast to the P2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac-(Gal3)Ser-Arg-pNa in comparison to Ac-Ser-Arg-pNa, and by the hydrolysis of Ac-(Glcalpha,beta)Tyr-Arg-pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P'4 glycosylated peptide [Abz-F-H-L-V-I-H-(GIcNAcbeta)N-E-EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K(m) and higher k(cat) values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P'4 and P4 glycosylated substrates.
我们合成了在P2或P3位点含有（Galβ）Ser或（Glcα,β）Tyr以及O - 乙酰化糖部分的短生色肽基 - Arg - 对硝基苯胺，并研究了它们被牛胰蛋白酶、木瓜蛋白酶、人组织激肽释放酶和大鼠托宁水解的情况。为了进行比较，我们检测了乙酰 - X - Arg - pNa和乙酰 - X - Phe - Arg - pNa系列（其中X为Ala、Phe、Gln和Asn）对这些酶的敏感性。我们还合成了具有人血管紧张素原氨基酸序列Phe8 - His - Leu - Val - Ile - His - Asn14的内部猝灭荧光肽，其中在Phe8之前和/或His13之后引入了[GlcNAcβ]Asn，并且在N端和C端连接了邻氨基苯甲酸（Abz）和N - [2,4 - 二硝基苯基] - 乙二胺（EDDnp）作为供体/受体荧光对。这些肽被检测作为人肾素、人组织蛋白酶D和猪胃蛋白酶的底物。带有亲水性糖部分的生色底物增加了它们对胰蛋白酶、组织激肽释放酶和大鼠托宁的敏感性。对于木瓜蛋白酶，糖的作用取决于其在底物中的位置，即在P3位置是不利的，与在P2位置导致亲和力增加相反，如Ac - （Gal3）Ser - Arg - pNa与Ac - Ser - Arg - pNa相比具有更高的抑制活性以及Ac - （Glcα,β）Tyr - Arg - pNa的水解所证明的那样。另一方面，糖羟基的乙酰化改善了除托宁外所有酶对敏感肽的水解。对应于血管紧张素原天然糖基化位点之一且P'4糖基化的肽[Abz - F - H - L - V - I - H - （GlcNAcβ）N - E - EDDnp]被证明是唯一对人肾素敏感的糖基化底物，并且与没有糖部分的相同肽相比，其水解时的K（m）值更低，k（cat）值更高。人组织蛋白酶D和猪胃蛋白酶对底物糖基化更耐受，能水解P'4和P4糖基化的底物。
