FLUOROGENIC SUBSTRATES FOR ACTIVATED PROTEIN-C - SUBSTRATE STRUCTURE-EFFICIENCY CORRELATION

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 aminonaphthalenesulfon amides (ANSN) as the detecting group and were substituted at P-4, P-3, P-2, 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 K-M values for APC below 100 mu M, reaching as low as 4-10 mu M 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 k(cat) exceeding 50 s(-1). These constants as well as k(cat)/K-M are influenced by the nature of amino acid in the P-3 and P-2 positions, by the isomer of ANSN (P'(1)), and by the structure of substituent incorporated in the sulfonamide moiety (P'(2)). The highest k(cat)/K-M were found for substrates with D-isomers of Leu, Phe, and Val in the P-3 position when these amino acids were N-unblocked. For the P-2 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 k(cat)/K-M. ANSN-containing substrates are hydrolyzed by both human and bovine APC at a similar rate. (C) 1995 Academic Press, Inc.