Design and synthesis of thrombin substrates with modified kinetic parameters

For the continuous registration of thrombin formation in plasma (1), selective thrombin substrates are required, that show moderate binding affinities (high K-m) and low turnover numbers (low k(cat)). Previously we have used SQ68 (CH3O-CO-CH2-CO-Aib-Arg-pNA) for this purpose. In order to find more substrates suitable for this application, we synthesized a series of 25 peptide p-nitroanilides. As lead structures SQ68 and S2238 (H-D-Phe-Pip-Arg-pNA) were used. By introduction of specific structure modifications we tried to alter the kinetic data in the required direction. The modifications were designed on basis of existing knowledge on the structure of the thrombin active-site and its surroundings. We indeed obtained a number of substrates with the kinetic constants in the desired range

Structural role of the tyrosine residues of cytochrome c

The tertiary structures of horse, tuna, Neurospora crassa, horse [Hse65,Leu67]- and horse [Hse65,Leu74]-cytochromes c were studied with high-resolution 1H n.m.r. spectroscopy. The amino acid sequences of these proteins differ at position 46, which is occupied by phenylalanine in the horse proteins but by tyrosine in the remaining two, and at positions 67, 74 and 97, which are all occupied by tyrosine residues in horse and tuna cytochrome c but in the other proteins are substituted by phenylalanine or leucine, though there is only one such substitution per protein. The various aromatic-amino-acid substitutions do not seriously affect the protein structure