Bordusa F, Ullmann D, Jakubke H D
Department of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
Biol Chem. 1997 Oct;378(10):1193-8.
The importance of electrostatic interactions between charged residues at the P3 position of substrates and the S3 subsite of the cysteine protease clostripain was investigated. For this purpose quantitative enzymatic hydrolysis studies using steady state kinetics have been carried out within a set of N alpha-protected synthetic dipeptide ester substrates with systematic changes of their charge in the P3 position. It was demonstrated that, in contrast to the former postulated second anionic S3 subsite, the lowest specificity was for the hydrolysis of the positively charged substrates. However, this effect was strongly dependent on the individual amino acid at P1. Furthermore, we investigated how far these P3-S3 interactions reflect on the S' subsite specificity via acyl transfers. Apart from the general weak influence of the charge at P3 on the deacylation kinetics, nucleophiles with proline at P'1 play an extraordinary role. Surprisingly, in contrast to the poor primary lysine specificity, acyl transfer using P1 lysine substrates does not affect the nucleophile efficiency found with the corresponding arginine substrates.
研究了底物P3位置的带电荷残基与半胱氨酸蛋白酶梭菌蛋白酶S3亚位点之间静电相互作用的重要性。为此，在一组Nα-保护的合成二肽酯底物中，通过稳态动力学进行了定量酶促水解研究，这些底物的P3位置电荷有系统变化。结果表明，与先前假定的第二个阴离子S3亚位点相反，带正电荷底物的水解特异性最低。然而，这种效应强烈依赖于P1位置的单个氨基酸。此外，我们研究了这些P3-S3相互作用通过酰基转移对S'亚位点特异性的影响程度。除了P3电荷对脱酰基动力学的一般微弱影响外，P'1位置为脯氨酸的亲核试剂起着特殊作用。令人惊讶的是，与初级赖氨酸特异性较差相反，使用P1赖氨酸底物的酰基转移并不影响相应精氨酸底物的亲核试剂效率。
