Pseudomonas aeruginosa alkaline proteinase might share a biological function with plasmin.
Pseudomonas aeruginosa alkaline proteinase, which is a zinc-dependent bacterial endopeptidase, preferentially hydrolyzed Boc-Val-Leu-Lys-methylcoumarylamide (MCA) which was originally designed as a specific substrate of plasmin, a plasma serine proteinase. The hydrolytic capacity was resistant to tosyl-lysine chloromethylketone at a concentration as high as 1 mM, but was blocked by a treatment with metal chelator such as o-phenanthroline at the concentration of 5 mM. Kinetic parameters of the amidolytic reaction were Km = 21 microM, kcat = 0.067 s-1 and kcat/Km = 3190 M-1 s-1. A synthetic peptide inhibitor which bore a possible ligand for zinc atom at the carboxy terminal was designed. This inhibitor, Ac-Val-Leu-Lys-4-mercaptoanilide, blocked the amidolytic activity of the pseudomonal alkaline proteinase in a competitive manner with the dissociation constant (Ki) value of 24 microM. The results imply that P. aeruginosa alkaline proteinase must be an unusual zinc-dependent 'C (COOH)-type' endopeptidase, which hydrolyzes the peptide bond of certain amino acid residues at the carboxyl group side by specific recognition, like serine- and cysteine-proteinases. In comparison, P. aeruginosa elastase which is a typical 'N (NH2)-type' metalloproteinase did not hydrolyze all of the commercially available peptide-MCA substrates tested at the present study. P. aeruginosa alkaline proteinase also hydrolyzed natural substrates of plasmin, such as fibrin and fibrinogen, with similar specific activities to plasmin. The susceptible subunits of fibrinogen were the A-alpha and B-beta ones, in this order. P. aeruginosa alkaline proteinase also exhibited an anti-coagulant activity in human plasma attributed to the direct fibrinogenolytic function. Such potential anti-coagulant capacity of the P. aeruginosa alkaline proteinase might explain, at least partly, the most characteristic pathologic feature of the P. aeruginosa septicemia, hemorrhagic lesions with lacking thrombi (Fetzer, A.E. et al. (1967) Am. Rev. Respirat. Dis. 96, 1121-1130).
Shibuya
Y
Y
Department of Laboratory Medicine, Kumamoto University Medical School, Japan.
Yamamoto
T
T
Morimoto
T
T
Nishino
N
N
Kambara
T
T
Okabe
H
H
D001777:Blood Coagulation; D010450:Endopeptidases / Q000502:physiology*; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000502:physiology*; D008666:Metalloendopeptidases / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D005337:Fibrin; D005340:Fibrinogen; D010450:Endopeptidases; D005341:Fibrinolysin; D008666:Metalloendopeptidases