title: Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.
abstract: A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular 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 characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.
authors.affiliation: Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
authors.forename: M
authors.initials: M
authors.lastname: Blaber
authors.forename: P J
authors.initials: PJ
authors.lastname: Isackson
authors.forename: J P
authors.initials: JP
authors.lastname: Burnier
authors.forename: J C
authors.initials: JC
authors.lastname: Marsters
authors.forename: R A
authors.initials: RA
authors.lastname: Bradshaw
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D007610:Kallikreins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D020840:Tissue Kallikreins
chemical_list: D009414:Nerve Growth Factors; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D007610:Kallikreins; C489120:Klk13 protein, mouse; D020840:Tissue Kallikreins; C489121:Klk1b22 protein, mouse; C489119:Klk9 protein, mouse; C053223:epidermal growth factor binding protein, mouse