McMurray J S, Budde R J, Ke S, Obeyesekere N U, Wang W, Ramdas L, Lewis C A
Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, 77030, USA.
Arch Biochem Biophys. 1998 Jul 1;355(1):124-30. doi: 10.1006/abbi.1998.0707.
A series of 48 cyclic peptides based on the amino acid sequence surrounding the autophosphorylation site of pp60(c-src) was synthesized and each was tested as both a substrate and an inhibitor of this protein tyrosine kinase. Starting with cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Ala6-Arg7-Gln8-d- Phe9-Pro10) a six-amino-acid survey was performed at positions 1 through 8 to determine which positions were critical for affinity and phosphorylation and which amino acids produced the greatest activity. Our survey found that Arg7 was detrimental for binding and phosphorylation and that aromatic residues were preferred at this position. Further increases in affinity were obtained with hydrophobic residues at position 6 with the optimum for both affinity and phosphorylation being Phe. Changes on the "amino-terminal" side of Tyr4 resulted in reduced Vmax values, illustrating the requirement for acidic residues in peptidic tyrosine kinase substrates. The result of the survey was cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Phe6-Phe7-Gln8-d-Phe 9-Pro10). The change of residues 6 and 7 resulted in a 42-fold increase in affinity and no increase in Vmax. As a substrate, this peptide displayed Michaelis-Menten kinetics at saturating ATP conditions. As an inhibitor, mixed inhibition was observed. A linear version of this peptide was 13-fold less potent an inhibitor than the cyclic peptide.
基于pp60(c-src)自身磷酸化位点周围氨基酸序列合成了一系列48种环肽，并将每种环肽作为该蛋白酪氨酸激酶的底物和抑制剂进行测试。从环（天冬氨酸1-天冬酰胺2-谷氨酰胺3-酪氨酸4-丙氨酸5-丙氨酸6-精氨酸7-谷氨酰胺8-d-苯丙氨酸9-脯氨酸10）开始，在第1至8位进行了六氨基酸研究，以确定哪些位置对亲和力和磷酸化至关重要，以及哪些氨基酸产生最大活性。我们的研究发现，精氨酸7不利于结合和磷酸化，该位置优选芳香族残基。在第6位带有疏水残基时亲和力进一步增加，亲和力和磷酸化的最佳残基均为苯丙氨酸。酪氨酸4“氨基末端”侧的变化导致最大反应速度值降低，这说明了肽类酪氨酸激酶底物中酸性残基的必要性。研究结果得到环（天冬氨酸1-天冬酰胺2-谷氨酰胺3-酪氨酸4-丙氨酸5-苯丙氨酸6-苯丙氨酸7-谷氨酰胺8-d-苯丙氨酸9-脯氨酸10）。第6和7位残基的变化使亲和力增加了42倍，而最大反应速度没有增加。作为底物，该肽在ATP饱和条件下呈现米氏动力学。作为抑制剂，观察到混合抑制作用。该肽的线性形式作为抑制剂的效力比环肽低13倍。
