General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease.
Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T-64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC). In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease. Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease. These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity. In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide.
Tang
Bing
B
Enzyme Laboratory, Biological Function Division, National Food Research Institute, Tsukuba, 305-8642, Ibaraki, Japan.
Nirasawa
Satoru
S
Kitaoka
Motomitsu
M
Marie-Claire
Cynthia
C
Hayashi
Kiyoshi
K
D000333:Aeromonas / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D002384:Catalysis; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; C073831:vibriolysin protein, Vibrio proteolyticus; C121413:PA protease protein, Aeromonas