Human plasma kallikrein. Preliminary studies on hydrolysis of proteins and peptides

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

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S1′ and S2′ subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

Biological Chemistry ◽

10.1515/bc.2008.166 ◽

2008 ◽

Vol 389 (12) ◽

Cited By ~ 6

Author(s):

Aurelio Resende Lima ◽

Fabiana M. Alves ◽

Pedro Francisco Ângelo ◽

Douglas Andrade ◽

Sachiko I. Blaber ◽

...

Keyword(s):

Amino Acids ◽

Human Plasma ◽

Receptor Agonist ◽

Plasma Kallikrein ◽

Tissue Kallikrein ◽

Charged Amino Acids ◽

B1 Receptor ◽

Lower Specificity ◽

Natural Amino Acids ◽

Hydrolysis Of

AbstractThe S1′ and S2′ subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO3H2)]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P1′ and P2′ positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS390S391RI-NH2was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S390and S391phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO3H2)391RI-NH2was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg9)-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S1′ subsite, with lower specificity for the S2′ subsite. Abz-MISLMKRPPGFSPFRSSRI-NH2was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO3H2) were poorly hydrolyzed. In conclusion, S1′ and S2′ subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.

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Studies on the temperature-dependent autoinhibition of human plasma kallikrein I

Biochemical Journal ◽

10.1042/bj2050529 ◽

1982 ◽

Vol 205 (3) ◽

pp. 529-534 ◽

Cited By ~ 4

Author(s):

P R Levison ◽

G Tomalin

Keyword(s):

Human Plasma ◽

Initial Velocity ◽

Sodium Deoxycholate ◽

Enzyme Concentration ◽

Low Molecular Weight ◽

Plasma Kallikrein ◽

Kinetic Effect ◽

Temperature Dependent ◽

Endogenous Inhibitors ◽

Hydrolysis Of

At 37 degrees C, human plasma kallikrein I follows Michaelis-Menten behaviour and exhibits a normal linear relationship between the initial velocity of hydrolysis of Ac-Pro-Phe-Arg-OMe, HCl and enzyme concentration in the range 0-150 pM. At temperatures of 30 degrees C and below substantial deviations from linearity are observed over the same enzyme concentration range. The temperature-dependent autoinhibition of kallikrein I activity is reversible and is not due to low-molecular-weight endogenous inhibitors or cofactors. The kinetic effect is apparently due to aggregation and can be abolished by the addition of sodium deoxycholate.

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The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein

Biochemical Journal ◽

10.1042/bj2030299 ◽

1982 ◽

Vol 203 (1) ◽

pp. 299-302 ◽

Cited By ~ 4

Author(s):

P R Levison ◽

G Tomalin

Keyword(s):

Human Plasma ◽

Methyl Esters ◽

Catalytic Efficiency ◽

Plasma Kallikrein ◽

Kinetics Of Hydrolysis ◽

Relative Differences ◽

Rate Limiting ◽

Kinetics Of ◽

Hydrolysis Of ◽

Limiting Values

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.

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Influence of Coagulation on the Activation of Plasminogen by Streptokinase and Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666939 ◽

1979 ◽

Vol 42 (03) ◽

pp. 901-908 ◽

Cited By ~ 25

Author(s):

Akikazu Takada ◽

Tetsumei Urano ◽

Yumiko Takada

Keyword(s):

Human Plasma ◽

Fibrin Clot ◽

Chromogenic Substrate ◽

Hydrolysis Of

SummaryHuman plasma was mixed with Ca++ or thrombin, and urokinase (UK) or streptokinase (SK) and a chromogenic substrate (S-2251: H-D-Val-Leu-Lys-pNA) specific to plasmin. The hydrolysis of S-2251 was higher when clot was formed by the addition of Ca++ or thrombin than in the absence of clot. The hydrolysis of S-2251 by euglobulin in the presence of UK was also higher when clot was formed, thus, inhibitors may not be related to the better activation of plasminogen, in the presence of fibrin clot. It may be suggested that plasminogen was better activated by activators (UK and SK) in the clot than in its absence.

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