In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64.
PA protease (pro-aminopeptidase processing protease) is an extracellular zinc metalloprotease produced by the Gram-negative bacterium Aeromonas caviae T-64. The 590-amino-acid precursor of PA protease is composed of a putative 19-amino-acid signal sequence, a 165-amino-acid N-terminal propeptide, a 33 kDa mature protease domain and an 11 kDa C-terminal propeptide. The proform of PA protease, which was produced as inclusion bodies in Escherichia coli, was subjected to in vitro refolding. It was revealed that the processing of the proform involved a stepwise autoprocessing mechanism. Firstly, the N-terminal propeptide was autocatalytically removed on completion of refolding and secondly, the C-terminal propeptide was autoprocessed after the degradation of the N-terminal propeptide. Both the N- and C-terminal propeptides existed as intact peptides after their successive removal, and they were subsequently degraded gradually. The degradation of the N-terminal propeptide appears to be the rate-limiting step in the maturation of the proform of PA protease.
Tang
Bing
B
Enzyme Laboratory, Biological Function Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
Nirasawa
Satoru
S
Kitaoka
Motomitsu
M
Hayashi
Kiyoshi
K
D000333:Aeromonas / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D004492:Edetic Acid; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D015032:Zinc
D001426:Bacterial Proteins; D011498:Protein Precursors; D004492:Edetic Acid; D010450:Endopeptidases; C121413:PA protease protein, Aeromonas; D015032:Zinc