scholarly journals Hydrolysis of somatostatin by human tissue kallikrein after the amino acid pair Phe-Phe

1997 ◽  
Vol 327 (1) ◽  
pp. 27-30 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Maria A. JULIANO ◽  
Luiz JULIANO
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Human Tissue ◽  
General Structure ◽  
Tissue Kallikrein ◽  
Growth Hormone Releasing Hormone ◽  
Amino Acid Pair ◽  
Reactive Centre Loop ◽  
Hydrolysis Of ◽  
Similar Kinetic

Somatostatin-(1–14) was hydrolysed by human tissue kallikrein at the Phe7-Trp8 bond, after a Phe-Phe pair of amino acids, with similar kinetic parameters to those described for human high- and low-molecular-mass kininogens. Substance P and human insulin, which also contain a Phe-Phe pair in their sequences, were both resistant. More details of this hydrolytic specificity of human tissue kallikrein were obtained by synthesizing and assaying internally quenched fluorescent peptides containing the sequence of somatostatin-(1–14), as well as the reactive-centre loop of human kallikrein-binding protein (kallistatin). We also observed that human tissue kallikrein hydrolysed growth hormone-releasing hormone at the Arg11-Lys12 bond, although this peptide contains in its structure a pair of leucines (Leu22-Leu23), in contrast with the Phe-Phe pair in somatostatin. We have also demonstrated the susceptibility to human tissue kallikrein of some chromogenic peptides with the general structure X-Phe-Phe-p-nitroanilide and of d-Pro-Phe-Phe-4-methylcoumaryl-7-amide.

scholarly journals Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids

1999 ◽  
Vol 339 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Julie CHAO ◽  
Lee CHAO ◽  
Maria A. JULIANO ◽  
Luiz JULIANO
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Tissue ◽  
Binding Protein ◽  
General Structure ◽  
Protein A ◽  
Tissue Kallikrein ◽  
Hybrid Peptides ◽  
Loop Sequence ◽  
Reactive Centre Loop

We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe–Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1–14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P´13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe–Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe–Arg and Leu–Lys bonds, respectively. Hybrid peptides containing kallistatin–PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P´-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe–Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe–Arg pair; however, hydrolysis after Phe–Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme–substrate interaction and/or particular substrate conformations.

scholarly journals Specificity of S'1 and S'2 subsites of human tissue kallikrein using the reactive-centre loop of kallistatin: the importance of P'1 and P'2 positions in design of inhibitors

2003 ◽  
Vol 371 (3) ◽  
pp. 1021-1025 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Sandro E. FOGAÇA ◽  
Robson L. MELO ◽  
Luiz JULIANO ◽  
Maria A. JULIANO
Keyword(s):  
Human Tissue ◽  
Serine Proteases ◽  
Potent Inhibitor ◽  
Competitive Inhibition ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Loop Sequence ◽  
Reactive Centre Loop ◽  
Hydrolysis Of ◽  
Peptide Analogues

We have demonstrated that the S´1 and S´2 subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. The presence of serine at position P´1 and arginine at P´2 resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P´1 and P´2 in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (Ki). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 inhibited hK1 in the range 20–30 nM (letters in italics denote the d-form of the amino acid). The peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 was a weak inhibitor for other serine proteases, as indicated by the higher Ki values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a Ki value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. In conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.

scholarly journals Specificity of human tissue kallikrein towards substrates containing Phe‒Phe pair of amino acids

1999 ◽  
Vol 339 (2) ◽  
pp. 473 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Julie CHAO ◽  
Lee CHAO ◽  
Maria A. JULIANO ◽  
Luiz JULIANO
Keyword(s):  
Amino Acids ◽  
Human Tissue ◽  
Tissue Kallikrein

Distribution of Growth Hormone-Releasing Hormone Like Immunoreactivity in Human Tissue Extracts*

1984 ◽  
Vol 59 (2) ◽  
pp. 263-268 ◽  
Author(s):  
TAMOTSU SHIBASAKI ◽  
YOSHINO KIYOSAWA ◽  
AKITSUGU MASUDA ◽  
MARI NAKAHARA ◽  
TOSHIHIRO IMAKI ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Human Tissue ◽  
Growth Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Tissue Extracts

scholarly journals Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments

1995 ◽  
Vol 312 (1) ◽  
pp. 233-238 ◽  
Author(s):  
E Del Nery ◽  
J R Chagas ◽  
M A Juliano ◽  
E S Prado ◽  
L Juliano
Keyword(s):  
Human Tissue ◽  
Kinetic Data ◽  
Time Course ◽  
Tissue Kallikrein ◽  
Hydrolysis Time ◽  
Porcine Tissue ◽  
Enzyme Substrate ◽  
Substrate Side ◽  
Kinetics Of ◽  
Hydrolysis Of

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P′4 to P′11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P′11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.

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.

Design of Inhibitors for Human Tissue Kallikrein Using Non-Natural Aromatic and Basic Amino Acids

2002 ◽  
Vol 383 (5) ◽  
pp. 853-857 ◽  
Author(s):  
Daniel C. Pimenta ◽  
Robson L. Melo ◽  
Giuseppe Caliendo ◽  
Vincenzo Santagada ◽  
Ferdinando Fiorino ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Human Tissue ◽  
Side Chains ◽  
Tissue Kallikrein ◽  
Urinary Kallikrein ◽  
Nonnatural Amino Acids ◽  
Basic Amino Acids ◽  
Competitive Inhibitors ◽  
Hydrophobic Amino Acids

Abstract We explored the unique substrate specificity of the primary S1 subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from PhenylacetylPheSer ArgEDDnp, a previously described inhibitor with analgesic and antiinflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099 1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by nonnatural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positivelycharged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the nonnatural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.

Human Tissue Kallikrein S1Subsite Recognition of Non-Natural Basic Amino Acids†

Biochemistry ◽  
2001 ◽  
Vol 40 (17) ◽  
pp. 5226-5232 ◽  
Author(s):  
Robson L. Melo ◽  
Roseli C. Barbosa Pozzo ◽  
Daniel C. Pimenta ◽  
Elisa Perissutti ◽  
Giuseppe Caliendo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Tissue ◽  
Tissue Kallikrein ◽  
Basic Amino Acids
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