Kinetic Peculiarities of Human Tissue Kallikrein: 1—Substrate Activation in the Catalyzed Hydrolysis of H--Valyl--leucyl--arginine 4-Nitroanilide and H--Valyl--leucyl--lysine 4-Nitroanilide; 2—Substrate Inhibition in the Catalyzed Hydrolysis of Nα-p-Tosyl--arginine Methyl Ester

2002 ◽  
Vol 400 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Marinez O. Sousa ◽  
Tânia L.S. Miranda ◽  
Caroline N. Maia ◽  
Eustáquio R. Bittar ◽  
Marcelo M. Santoro ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Human Tissue ◽  
Substrate Inhibition ◽  
Tissue Kallikrein ◽  
Substrate Activation ◽  
Hydrolysis Of

scholarly journals Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1974 ◽  
Vol 141 (2) ◽  
pp. 365-381 ◽  
Author(s):  
Christopher W. Wharton ◽  
Athel Cornish-Bowden ◽  
Keith Brocklehurst ◽  
Eric M. Crook
Keyword(s):  
Methyl Ester ◽  
Dissociation Constant ◽  
Computational Methods ◽  
Rate Constant ◽  
Guanidine Hydrochloride ◽  
Ester Hydrolysis ◽  
Binary Complex ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Ester Substrate

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10-2±0.32×10-2s-1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s-1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s-1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

scholarly journals Substrate Inhibition in the Hydrolysis of Hippuric Acid Esters by Carboxypeptidase A

1975 ◽  
Vol 53 (2) ◽  
pp. 283-294 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Substrate Concentration ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Side Chain ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Acid Esters

The dependence of initial velocity upon substrate concentration has been examined in the carboxypeptidase A catalyzed hydrolysis of the following hippuric acid esters (at pH 7.5, 25°, ionic strength O.5): C6H5CONHCH2CO2CHRCO2H: R=CH3; CH2CH3;(CH2)2CH3; (CH2)3CH3; (CH2)5CH3; CH(CH3)2; CH2CH(CH3)2; C6H5; CH2C6H5. All of these esters display marked substrate inhibition of their enzymic hydrolyses. With the exception of R=CH3, the velocity-substrate concentration profiles for each of these esters can be rationalized by the formation of an E.S2 complex which, independent of the alcohol moiety of the ester, reacts approximately 25 times more slowly than the E.S complex. For most of these esters, the formation of E.S2 approximates ordered binding of the substrate molecules at the catalytic and inhibitory sites. While binding at the catalytic site is markedly dependent on the nature of the R group, binding of a second substrate molecule to E.S is not significantly affected by the nature of the R side chain. For R=C6H5, the D ester is neither a substrate nor a competitive inhibitor of the hydrolysis of the L-ester but can replace the L-ester at the binding site which is responsible for substrate inhibition. The kinetic analysis suggests that this behavior of D and L -enantiomers is also typical of the other esters examined (except possibly R=CH3). For R=CH3 only, substrate activation also seems to occur prior to the onset of substrate inhibition at higher substrate concentrations.

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.

Substrate Activation in the Carboxypeptidase A Catalysis of Ester Hydrolysis

1974 ◽  
Vol 52 (23) ◽  
pp. 3829-3836 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Phenyl Ring ◽  
Mandelic Acid ◽  
Substrate Inhibition ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Derivatives Of ◽  
Substrate Concentrations ◽  
Acid Unit ◽  
Reaction Schemes

The hydrolyses of the O-hippuryl derivatives of glycolic acid (1a), 2-methyllactic acid (1b), and p-chloromandelic acid (1c) by bovine carboxypeptidase A display substrate activation. The hydrolyses of the latter two esters also display substrate inhibition at high substrate concentrations (>0.03 and >0.05 M respectively). Partial kinetic analyses are presented, and these phenomena are discussed in terms of reaction schemes which involve substrate binding at both activating and inhibiting regulatory sites.The hydrolysis of 1b by this enzyme is the first indication that the presence of a hydrogen atom on the α-carbon atom of the alcohol moiety is not obligatory for ester substrates of carboxypeptidase A. The binding of 1c at the catalytic site is approximately 1000 times weaker than for O-hippurylmandelic acid and indicates a dramatic influence for the p-chloro substituent on the binding of the phenyl ring of the mandelic acid unit.

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.

Further studies of the specificity of carboxypeptidase A towards hippuric acid esters

1978 ◽  
Vol 56 (16) ◽  
pp. 2188-2193
Author(s):  
John W. Bunting ◽  
Samuel S.-T. Chu
Keyword(s):  
Ionic Strength ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Carboxypeptidase A ◽  
Hydrophobic Pocket ◽  
Substrate Activation ◽  
Kinetics Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Acid Esters

The kinetics of hydrolysis of a series of 10 new hippurate esters (C6H5CONHCH2CO2CRR1CO2H (I)) by bovine pancreatic carboxypeptidase A have been investigated at pH 7.5, 25 °C, and ionic strength 0.5. Pronounced substrate inhibition was displayed by I: R = H, R1 = C6H5(CH2)2, 3-indolylmethyl, 4-HOC6H4CH2, and 4-FC6H4 whereas pronounced substrate activation was observed for I: R = H, R1 = 4-CH3C6H4, 4-C2H5C6H4, 4-C6H5C6H4, 1-naphthyl, 2-naphthyl, and R = R1 = C2H5. In all cases substrate activation and substrate inhibition were shown to be consistent with ES2 complex formation similar to that previously observed for other hippurate esters. Kinetic parameters were evaluated for each ester and it is noted that ail 13 hippurate esters now known to display substrate inhibition have kcat/Km > 106 M−1 min−1, whereas kcat/km < 106 M−1 min−1 for all 9 hippurate esters known to display substrate activation. The enzymic specificity for the R1 unit of I suggests binding of R1 in a 'bent' hydrophobic pocket having a restricted entrance.

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.

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