Butenas S, Ribarik N, Mann K G
Department of Biochemistry, University of Vermont, Burlington 05405.
Biochemistry. 1993 Jul 6;32(26):6531-8. doi: 10.1021/bi00077a006.
A series of 100 tripeptide fluorogenic substrates has been synthesized. These substrates contain Arg in the P1 position, various amino acids in the P2 and P3 positions, and different 6-amino-1-naphthalenesulfonamides (ANSN) as the detecting group (P'). The 38 compounds possessing the highest initial rates of factor VIIa hydrolysis were evaluated for substrate kinetic parameters in the presence and absence of tissue factor (TF) and by factor Xa. Most of these substrates had a higher kcat/KM (keff) value for the factor VIIa-TF complex than for factor Xa. Substitution of different amino acids in the P2 position showed that substrates with bulkier amino acids such as Leu, Pro, and Val have higher values for KM and kcat than those with smaller amino acids (Gly or Ser). The highest second-order rate constants were found for substrates with Val or Pro in the P2 position. A decrease or increase in volume of the P2 substituent (Gly, Ser, or Leu) resulted in a decrease in this constant. Substrates with the highest keff values have Phe in the P3 position. As the hydrophobicity and volume of the amino acid in the P3 position decreased, the keff was reduced. The efficiency of substrates for hydrolysis by factor VIIa was enhanced by an increase of hydrophobicity in the P' structure. TF enhanced the amidolytic activity of the "family" of 38 substrates with ANSN in the P' position on an average of 58-fold.
已合成了一系列100种三肽荧光底物。这些底物在P1位置含有精氨酸，在P2和P3位置含有各种氨基酸，并含有不同的6-氨基-1-萘磺酰胺（ANSN）作为检测基团（P'）。对38种具有最高因子VIIa水解初始速率的化合物，在有和没有组织因子（TF）的情况下以及通过因子Xa评估其底物动力学参数。这些底物中的大多数对于因子VIIa-TF复合物的kcat/KM（keff）值高于因子Xa。在P2位置替换不同的氨基酸表明，具有较大氨基酸（如亮氨酸、脯氨酸和缬氨酸）的底物比具有较小氨基酸（甘氨酸或丝氨酸）的底物具有更高的KM和kcat值。在P2位置含有缬氨酸或脯氨酸的底物具有最高的二级速率常数。P2取代基（甘氨酸、丝氨酸或亮氨酸）体积的减小或增加导致该常数降低。具有最高keff值的底物在P3位置含有苯丙氨酸。随着P3位置氨基酸的疏水性和体积减小，keff降低。P'结构中疏水性的增加提高了底物被因子VIIa水解的效率。TF使在P'位置含有ANSN的38种底物“家族”的酰胺水解活性平均提高了58倍。
