scholarly journals Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases

1995 ◽  
Vol 311 (1) ◽  
pp. 275-282 ◽  
Author(s):  
E Pol ◽  
S L Olsson ◽  
S Estrada ◽  
T W Prasthofer ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Active Site ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Association Rate ◽  
Dissociation Equilibrium ◽  
Cystatin A ◽  
Association Rate Constants ◽  
Human Cystatin ◽  
Chicken Cystatin

The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the ‘occluding loop’ of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.

scholarly journals Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin

1992 ◽  
Vol 281 (1) ◽  
pp. 49-55 ◽  
Author(s):  
P Lindahl ◽  
M Abrahamson ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Rate Constants ◽  
Proteinase Inhibitors ◽  
Equilibrium Constants ◽  
Fluorescence Emission ◽  
Cysteine Proteinases ◽  
Human Cystatin C ◽  
Kinetics Of ◽  
Human Cystatin ◽  
Chicken Cystatin

The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s-1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine-proteinase inhibitors and their target enzymes. The model for the proteinase-inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C.

scholarly journals Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes

1994 ◽  
Vol 299 (1) ◽  
pp. 219-225 ◽  
Author(s):  
I Björk ◽  
E Pol ◽  
E Raub-Segall ◽  
M Abrahamson ◽  
A D Rowan ◽  
...  
Keyword(s):  
Rate Constant ◽  
Cystatin C ◽  
Rate Constants ◽  
Dissociation Rate ◽  
Dissociation Rate Constant ◽  
Association Rate Constant ◽  
Association Rate ◽  
Human Cystatin C ◽  
Human Cystatin ◽  
Chicken Cystatin

The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.

scholarly journals Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations

1991 ◽  
Vol 276 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P Lindahl ◽  
E Raub-Segall ◽  
S T Olson ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Equilibrium Constants ◽  
Sulphonic Acid ◽  
Cysteine Proteinases ◽  
Human Cystatin C ◽  
Human Cystatin ◽  
Chicken Cystatin

Papain was labelled by attachment of the fluorescent groups 2-(4′-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.

scholarly journals Effect of heparin on the glia-derived-nexin-thrombin interaction

1989 ◽  
Vol 257 (1) ◽  
pp. 191-196 ◽  
Author(s):  
A Wallace ◽  
G Rovelli ◽  
J Hofsteenge ◽  
S R Stone
Keyword(s):  
Rate Constants ◽  
Antithrombin Iii ◽  
Kinetic Properties ◽  
Association Rate ◽  
High Affinity ◽  
Diffusion Controlled ◽  
Different Types ◽  
Human Thrombin ◽  
Alpha Beta ◽  
Association Rate Constants

In order to determine the specificity of the interaction between thrombin and glia-derived nexin (GdN), the inactivation of proteolytically modified human thrombin species by GdN has been studied. The second-order rate constants for the inactivation of alpha-, beta T-, gamma T- and epsilon-thrombin by GdN were 1.41, 0.63, 0.33 and 1.91 microM-1.s-1 respectively. The kinetic properties of gdN were also investigated in the presence of different types of heparin, fractionated according to antithrombin III-binding affinity. Association rate constants of both gdN and antithrombin III with alpha-thrombin were obtained using unfractionated, low- and high-affinity heparin types. The different heparin types gave optimal rates of inhibition at similar heparin concentrations for both inhibitors. At optimal heparin concentrations, the rate of inactivation of alpha-thrombin by GdN was 0.5-1.2 nM-1.s-1, which suggests that, under these conditions, the interaction is diffusion-controlled.

scholarly journals Association Rate Constants as Determinants of Ligand Selectivity:Lessons from The Dopamine And The Serotonin Transporter

2015 ◽  
Vol 29 (S1) ◽  
Author(s):  
Michael Freissmuth ◽  
Peter Hasenhuetl ◽  
Sonja Sucic ◽  
Harald Sitte ◽  
Walter Sandtner
Keyword(s):  
Serotonin Transporter ◽  
Rate Constants ◽  
Association Rate ◽  
Association Rate Constants
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