Butenas S, Drungilaite V, Mann K G
Department of Biochemistry, University of Vermont, Burlington 05405, USA.
Anal Biochem. 1995 Mar 1;225(2):231-41. doi: 10.1006/abio.1995.1148.
A series of 87 fluorescent peptide substrates have been synthesized and evaluated using human and bovine activated protein C (APC). These substrates contain various isomers of aminonaphthalenesulfonamides (ANSN) as the detecting group and were substituted at P4, P3, P2, P'1, and P'2 positions. Substrates with 6,2-ANSN at P'1 had the highest fluorescence quantum yield, exceeding that of 6,1-ANSN 9.3-fold and 5,1-ANSN almost 1000-fold. Almost 50 substrates containing substituted ANSNs as leaving groups have KM values for APC below 100 microM, reaching as low as 4-10 microM for 8 of these substrates. These values are significantly lower than those reported for p-nitroanilide and 4-methylcoumaryl-7-amide substrates. Additionally, some of these substrates have relatively high kcat exceeding 50 s-1. These constants as well as kcat/KM are influenced by the nature of amino acid in the P3 and P2 positions, by the isomer of ANSN (P'1), and by the structure of substituent incorporated in the sulfonamide moiety (P'2). The highest kcat/KM were found for substrates with D-isomers of Leu, Phe, and Val in the P3 position when these amino acids were N-unblocked. For the P2 position Val, Phe, and Leu were preferable. Substrates containing n-butyl (bovine APC) and benzyl (human APC) substituents in the (P'2) structure have elevated kcat/KM. ANSN-containing substrates are hydrolzyed by both human and bovine APC at a similar rate.
已合成了一系列87种荧光肽底物，并使用人源和牛源活化蛋白C（APC）进行了评估。这些底物包含各种氨基萘磺酰胺（ANSN）异构体作为检测基团，并在P4、P3、P2、P'1和P'2位置进行了取代。P'1位置带有6,2 - ANSN的底物具有最高的荧光量子产率，比6,1 - ANSN高9.3倍，比5,1 - ANSN几乎高1000倍。近50种含有取代ANSN作为离去基团的底物对APC的KM值低于100 microM，其中8种底物的KM值低至4 - 10 microM。这些值显著低于对硝基苯胺和4 - 甲基香豆素 - 7 - 酰胺底物所报道的值。此外，其中一些底物具有相对较高的kcat，超过50 s-1。这些常数以及kcat/KM受P3和P2位置氨基酸的性质、ANSN（P'1）的异构体以及磺酰胺部分（P'2）中引入的取代基结构的影响。当P3位置的氨基酸N未被封闭时，P3位置具有Leu、Phe和Val的D - 异构体的底物具有最高的kcat/KM。对于P2位置，Val、Phe和Leu是优选的。在（P'2）结构中含有正丁基（牛APC）和苄基（人APC）取代基的底物具有升高的kcat/KM。含ANSN的底物被人源和牛源APC以相似的速率水解。
