Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bond of prekallikrein in the activation.
Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23-27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg371-Ile372 bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Arg-Ile-Val-4- nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of Km = 118 microM, kcat = 1.56/s and kcat/Km = 1.33.10(4)/s M. These results indicated that the Arg371-Ile372 bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by pseudomonal elastase, however, revealed that the activation rate was slow, though the Km values was good enough to expect an occurrence of this activation in vivo (Km = 248 nM, kcat = 6.8.10(-4)/s, and kcat/Km = 2.7.10(3)/s M). The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biological significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.
Tanaka
H
H
First Department of Surgery, Kumamoto University Medical School, Japan.
Yamamoto
T
T
Shibuya
Y
Y
Nishino
N
N
Tanase
S
S
Miyauchi
Y
Y
Kambara
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D005174:Factor XII / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011288:Prekallikrein / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D005174:Factor XII; D011288:Prekallikrein; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa