title: Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase.
abstract: An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.
authors.affiliation: Applied Enzymology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Nirasawa
authors.forename: Y
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Z Z
authors.initials: ZZ
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Yoshida
authors.forename: K
authors.initials: K
authors.lastname: Hayashi
mesh_terms: D000333:Aeromonas / Q000201:enzymology*; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D004789:Enzyme Activation; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D011489:Protein Denaturation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014508:Urea; D015032:Zinc
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011480:Protease Inhibitors; D011498:Protein Precursors; D014508:Urea; D000626:Aminopeptidases; C038222:bacterial leucyl aminopeptidase; D008666:Metalloendopeptidases; D015032:Zinc