Nerve growth factor alpha subunit: effect of site-directed mutations on catalytic activity and 7S NGF complex formation.
Mouse alpha- and gamma-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with beta-NGF. gamma-NGF is an active arginine-specific esteropeptidase; the alpha-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of alpha-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 gamma-NGF. Seven of the 15 derivatives that remained more alpha-like were able to substitute for native alpha-NGF in reforming 7S complexes; the other eight derivatives that were more gamma-like showed greatly reduced ability to do so. However, the most gamma-like alpha-NGF derivative could not substitute for native gamma-NGF in 7S complex formation. These findings suggest that the alpha-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.
Yarski
M A
MA
Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, CA 92697, USA.
Bax
B D
BD
Hogue-Angeletti
R A
RA
Bradshaw
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000737:chemistry*; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020932:Nerve Growth Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D009414:Nerve Growth Factors / Q000737:chemistry; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013363:Submandibular Gland / Q000201:enzymology
C013614:7S nerve growth factor complex; C488871:7S nerve growth factor protein, human; D004792:Enzyme Precursors; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D020932:Nerve Growth Factor; D010450:Endopeptidases