title: Substrate specificity of a hypothalamic neurosecretory granule enzyme capable of processing pro-gonadotropin releasing hormone precursor protein.
abstract: We examined the specificity of a bovine hypothalamic neurosecretory granule enzyme which we discovered and which is capable of processing pro-gonadotropin releasing hormone precursor protein to yield gonadotropin associated peptide and a C-terminal extended form of gonadotropin releasing hormone (Palen et al.). The sequence in the precursor protein that separates the two active peptides is -Gly-Gly-Lys-Arg- where the pair of basic residues, -Lys-Arg-, is the anticipated cleavage site. On the basis of Vmax/Km as the measure of substrate specificity, Benzoyl(Bz)-Gly-Gly-Lys-Arg-2-Napthylamide (NA) greater than Bz-Gly-Gly-Arg-Lys-2-NA much greater than Bz-Gly-Gly-Arg-Arg-2-NA approximately equal to Bz-Gly-Gly-Lys-Lys-2-NA. Bz-Gly-Gly-Lys(N(epsilon)-acetyl)-Arg-2-NA is a very poor substrate. Our results indicate that the composition and sequence of the pair of basic residues at the primary cleavage site is important for enzyme specificity and that changes in the P1 or P2 residues of a potential substrate may affect both Km and Vmax. Hydrolysis of all substrates occurs at the P1-2-NA bond. We had previously shown that there is no cleavage between the pair of basic residues. With longer peptide substrates, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA greater than Bz-Gly-Leu-Arg(NO2)-Pro-Gly-Gly-Lys-Arg-2-NA greater than Bz-Gly-Gly-Lys-Arg-2-NA. Extending the substrate sequence to more closely resemble the amino acid sequence in the precursor protein improves Km 10-fold and Vmax about 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009490:Neurosecretory Systems / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D010450:Endopeptidases; C052441:gonadotropin-associated-peptide releasing enzyme