SYNTHETIC CHIMERAS OF MOUSE GROWTH FACTOR-ASSOCIATED GLANDULAR KALLIKREINS .1. KINETIC-PROPERTIES

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glan dular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF) - were expressed in Escherichia coli and isolated, and their kinetic properties were c haracterized. The assembly of these synthetic proteases involved the s ubstitution of regions of the proteins containing four specific surfac e loops that have been postulated to influence both kinetic specificit y and the formation of growth factor complexes. The substrates utilize d in the kinetic characterization of these chimeric kallikreins were t ripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kall ikreins in the precursors. Characterization of these hybrid enzymes de monstrates that K(m) and k(cat) kinetic constants may be independently affected by the regions utilized in construction of these chimeric ka llikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enh anced the k(cat) for substrates containing threonine in the P2 positio n, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins cont aining loop 2 and the kallikrein loop dictated the generally inverted K(m) and k(cat) kinetic constants observed between EGF-BP and gamma-NG F. Finally, in gamma-NGF the autolysis loop, found in the carboxyl ter minal region, functions to lower the K(m) kinetic constant for a varie ty of substrates. The results allow previously characterized kinetic d ifferences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid p ositions responsible for these functional characteristics.