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

2008 ◽  
Vol 389 (12) ◽  
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.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

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.

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

1978 ◽  
Vol 8 (1-2) ◽  
pp. 125-131 ◽  
Author(s):  
Claudio A. M. Sampaio ◽  
Daisy Grisolia
Keyword(s):  
Human Plasma ◽  
Plasma Kallikrein ◽  
Hydrolysis Of ◽  
Proteins And Peptides

scholarly journals Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1431-1436
Author(s):  
CF Scott ◽  
HR Wenzel ◽  
HR Tschesche ◽  
RW Colman
Keyword(s):  
Human Plasma ◽  
Serine Proteases ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Disease States ◽  
Reactive Center ◽  
Experimental Drug ◽  
Factor Xiia ◽  
Factor Xia ◽  
Altered Form

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)- 1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(- 8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine- directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15- aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

scholarly journals An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles

2008 ◽  
Vol 415 (3) ◽  
pp. 401-407 ◽  
Author(s):  
Soo-Jin Yeom ◽  
Hye-Jung Kim ◽  
Jung-Kul Lee ◽  
Dong-Eun Kim ◽  
Deok-Kun Oh
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hydrophobic Interactions ◽  
Electron System ◽  
Rhodococcus Rhodochrous ◽  
Aromatic Nitriles ◽  
Charged Amino Acids ◽  
Aromatic Substrates ◽  
In The Wild ◽  
Hydrolysis Of

Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated π-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity.

Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds

1999 ◽  
Vol 45 (1-3) ◽  
pp. 145-149 ◽  
Author(s):  
Maria Luiza V Oliva ◽  
Sonia Andrade ◽  
Isabel F.C Batista ◽  
Misako U Sampaio ◽  
Maria Juliano ◽  
...  
Keyword(s):  
Human Plasma ◽  
Factor Xa ◽  
Plasma Kallikrein ◽  
Factor Xa Inhibitor ◽  
Tissue Kallikrein ◽  
Reactive Site

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

1982 ◽  
Vol 205 (3) ◽  
pp. 529-534 ◽  
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.

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

1982 ◽  
Vol 203 (1) ◽  
pp. 299-302 ◽  
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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