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 y-subunit of nerve growth factor (7-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 @-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that K, and kc,, 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, 1983), in y-NGF enhanced the kc,, for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the 0-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted K , and k, , kinetic constants observed between EGF-BP and y-NGF. Finally, in y-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the K, kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and y-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.

Prediction of the three‐dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein

“…The relationship of this numbering scheme to that used by Blaber et al (1989Blaber et al ( , 1993a) is described in the legend to Figure I . N-terminal sequence ( Fig.…”

“…Numbers in parentheses-(AI), (B3), etc. -indicate the equivalent residue number in the scheme used by Blaber et al (1989Blaber et al ( , 1993a, in which each of the three chains in the mature forms of y-NGF and EGF-BP is numbered sequentially. In y-NGF four residues cleaved from the kallikrein loop (residues 95f, 95g, 95h, and 9%) the second chain starts at residue Phe 9Sj (Bl); the cleavage in the autocatalytic loop occurs after Lys 148; the first residue in the third chain is Phe 149 (Cl).…”

Synthetic chimeras of mouse growth factor‐associated glandular kallikreins. II. Growth factor binding properties

“…Thus, the y-subunit carboxyl terminal region appears to have improved the binding affinity within the substrate binding pocket, which apparently has compensated for the increase in K , values associated with the nonglycosylated r-EGF-BP. The substitution of region 1 of EGF-BP with y-NGF has no effect upon the catalysis of a small EGF-like substrates (which probes substrate S3 to SI contacts; Blaber et al, 1993). Thus, region 1 of EGF-BP, which is required for complex formation, also interacts with EGF outside of the S3 to SI positions.…”