Substrate specificities of growth factor associated kallikreins of the mouse submandibular gland

Abstract

The kinetic constants for the hydrolysis of a series of tripeptide p-nitroanilide substrates by mouse epidermal growth factor binding protein (EGF-BP), the .gamma.-subunit of mouse nerve growth factor (.gamma.-NGF), bovine pancreatic trypsin (BPT), and porcine pancreatic kallikrein (PPK) have been evaluated. These substrates correspond to the carboxyl-terminal three amino acids of the mature forms of epidermal growth factor (EGF) and .beta.-nerve growth factor (.beta.-NGF), as well as various substitutions in the penultimate and antepenultimate positions, and, as such, represent potential recognition sites for precursor processing. The mouse kallikreins (EGF-BP and .gamma.-NGF) preferentially hydrolyze the substrates with the sequences of their specifically associated growth factors; however, the constants derived from these reactions do not account for the association constants observed with the mature growth factors, and additional significant binding interactions between EGF-BP and EGF and between .gamma.-NGF and .beta.-NGF are predicted to exist outside of the catalytic binding site, i.e., the P3 to P1 positions. A comparison of the kinetic constants of BPT, PPK, and the mouse kallikreins indicates that EGF-BP and .gamma.-NGF display a hybrid catalytic character. A favorable substrate P1 arginine guanidinium group interaction exits for the mouse kallikreins, similar to that of BPT, but a preference for a hydrophobic side chain in the substrate P2 position makes the mouse kallikreins, especially EGF-BP, more closely resemble PPK than BPT. These findings have significant implications with regard to molecular modeling of the mouse kallikreins.