Nerve growth factor α subunit: effect of site-directed mutations on catalytic activity and 7S NGF complex formation

• Published in: Biochimica et biophysica acta Vol. 1477; no. 1; pp. 253 - 266
• Main Authors: Yarski, Michael A, Bax, Ben D, Hogue-Angeletti, Ruth A, Bradshaw, Ralph A
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 07.03.2000
• Subjects: Amino Acid Sequence; Animals; Catalysis; Cell Line; Chromatography, Gel; Conformation; Endopeptidases - chemistry; Enzyme Precursors - chemistry; Humans; Kallikrein; Mass Spectrometry; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nerve Growth Factor - biosynthesis; Nerve Growth Factor - chemistry; Nerve Growth Factor - genetics; Nerve Growth Factors - chemistry; Plasmids; Protein Conformation; Recombinant Proteins - chemistry; Serine protease; Submandibular Gland - enzymology; Zymogen; Zymogen; Kallikrein; Serine protease; Mass spectrometry; Conformation
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(99)00277-0

Mouse α- and γ-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with β-NGF. γ-NGF is an active arginine-specific esteropeptidase; the α-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of α-NGF to alter the N-terminus and three residues in loop 7, a region that contributes to the catalytic center, restored substantial catalytic activity against N-benzoyl arginine- p-nitroanilide as substrate in two derivatives although they were not as active as recombinant γ-NGF. Seven of the 15 derivatives that remained more α-like were able to substitute for native α-NGF in reforming 7S complexes; the other eight derivatives that were more γ-like showed greatly reduced ability to do so. However, the most γ-like α-NGF derivative could not substitute for native γ-NGF in 7S complex formation. These findings suggest that the α-NGF backbone can be corrected to a functional enzyme by the addition of a normal N-terminal structure and two catalytic site substitutions and that the 7S complex requires one kallikrein subunit in the zymogen form and one in an active conformation.