scholarly journals Interaction of chicken cystatin with inactivated papains

1989 ◽  
Vol 260 (1) ◽  
pp. 61-68 ◽  
Author(s):  
I Björk ◽  
K Ylinenjärvi
Keyword(s):  
Rate Constants ◽  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Tight Binding ◽  
Cysteine Residue ◽  
Dissociation Rate ◽  
Covalent Attachment ◽  
Active Site Cysteine ◽  
High Affinity ◽  
Chicken Cystatin

Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd less than approximately 15 pM), although less tightly than did active papain (Kd approximately 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido-(4-guanido)butane were 0.17 and approximately 10 microM respectively. The spectroscopic changes accompanying the reaction of all but the most weakly binding (Kd greater than or equal to 2 microM) inactivated papains with cystatin were similar to those induced by the active enzyme. Interactions involving the reactive cysteine residue of papain are thus not crucial for high-affinity binding of the enzyme to cystatin, in accordance with a recently proposed model for the enzyme-inhibitor complex, based on computer docking experiments. In this model there is sufficient space around the reactive cysteine in the complex for a small inactivating group, explaining the tight binding of papains with such substituents. However, larger inactivating groups cannot be accommodated in this space and therefore must displace the inhibitor out of the tight fit with the enzyme, in agreement with the observed decrease in binding affinity with increasing size of bulkier substituents. The kinetics of binding of cystatin to inactivated papains were compatible with simple, reversible, bimolecular reactions, having association rate constants of (7-9) x 10(6) M-1 s-1 at pH 7.4, 25 degrees C, similar to what was shown previously for the binding of cystatin to active papain. The rate of association of the inhibitor with either active or inactivated papain thus appears to be primarily diffusion-controlled. The decreasing affinity of cystatin for papains inactivated with groups of increasing size was shown to be due to progressively higher dissociation rate constants, consistent with the greater impairment of fit between the binding regions of the two molecules.

scholarly journals Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.

1980 ◽  
Vol 84 (1) ◽  
pp. 141-150 ◽  
Author(s):  
L G Bergen ◽  
G G Borisy
Keyword(s):  
Electron Microscope ◽  
Rate Constants ◽  
Dissociation Constants ◽  
Dissociation Rate ◽  
Association Constants ◽  
Directional Growth ◽  
Microtubule Protein ◽  
Electron Microscope Analysis ◽  
Dissociation Rate Constants

Microtubules are polar structures, and this polarity is reflected in their biased directional growth. Following a convention established previously (G. G. Borisy, 1978, J. Mol. Biol. 124:565--570), we define the plus (+) and minus (-) ends of a microtubule as those equivalent in structural orientation to the distal and proximal ends, respectively, of the A subfiber of flagellar outer doublets. Rates of elongation were obtained for both ends using flagellar axonemes as seeds and porcine brain microtubule protein as subunits. Since the two ends of a flagellar seed are distinguishable morphologically, elongation of each end may be analyzed separately. By plotting rates of elongation at various concentrations of subunit protein, we have determined the association and dissociation rate constants for the plus and minus ends. Under our conditions at 30 degrees C, the association constants were 7.2 X 10(6) M-1 s-1 and 2.25 X 10(6) M-1 s-1 for the plus and minus ends, respectively, and the dissociation constants were 17 s-1 and 7 s-1. From these values and Wegner's equations (1976, J. Mol. Biol. 108:139--150), we identified the plus end of the microtubule as its head and calculated "s," the head-to-tail polymerization parameter. Surprisingly small values (s = 0.07 +/- 0.02) were found. The validity of models of mitosis based upon head-to-tail polymerization (Margolis et al., 1978, Nature (Lond.) 272:450--452) are discussed in light of a small value for s.

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 Slow-tight-binding inhibition of enoyl-acyl carrier protein reductase from Plasmodium falciparum by triclosan

2004 ◽  
Vol 381 (3) ◽  
pp. 719-724 ◽  
Author(s):  
Mili KAPOOR ◽  
C. Chandramouli REDDY ◽  
M. V. KRISHNASASTRY ◽  
Namita SUROLIA ◽  
Avadhesha SUROLIA
Keyword(s):  
Plasmodium Falciparum ◽  
Fatty Acid ◽  
Rate Constants ◽  
Enzyme Inhibitor ◽  
Acyl Carrier Protein ◽  
Tight Binding ◽  
Carrier Protein ◽  
Type I ◽  
Type Ii ◽  
Inhibitor Complex

Triclosan is a potent inhibitor of FabI (enoyl-ACP reductase, where ACP stands for acyl carrier protein), which catalyses the last step in a sequence of four reactions that is repeated many times with each elongation step in the type II fatty acid biosynthesis pathway. The malarial parasite Plasmodium falciparum also harbours the genes and is capable of synthesizing fatty acids by utilizing the enzymes of type II FAS (fatty acid synthase). The basic differences in the enzymes of type I FAS, present in humans, and type II FAS, present in Plasmodium, make the enzymes of this pathway a good target for antimalarials. The steady-state kinetics revealed time-dependent inhibition of FabI by triclosan, demonstrating that triclosan is a slow-tight-binding inhibitor of FabI. The inhibition followed a rapid equilibrium step to form a reversible enzyme–inhibitor complex (EI) that isomerizes to a second enzyme–inhibitor complex (EI*), which dissociates at a very slow rate. The rate constants for the isomerization of EI to EI* and the dissociation of EI* were 5.49×10−2 and 1×10−4 s−1 respectively. The Ki value for the formation of the EI complex was 53 nM and the overall inhibition constant Ki* was 96 pM. The results match well with the rate constants derived independently from fluorescence analysis of the interaction of FabI and triclosan, as well as those obtained by surface plasmon resonance studies [Kapoor, Mukhi, N. Surolia, Sugunda and A. Surolia (2004) Biochem. J. 381, 725–733].

scholarly journals Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin

1992 ◽  
Vol 286 (1) ◽  
pp. 165-171 ◽  
Author(s):  
P Lindahl ◽  
M Nycander ◽  
K Ylinenjärvi ◽  
E Pol ◽  
I Björk
Keyword(s):  
Rate Constants ◽  
Fluorescence Emission ◽  
Terminal Segment ◽  
Dissociation Rate ◽  
Binding Energies ◽  
Terminal Region ◽  
Cysteine Proteinases ◽  
Difference Spectra ◽  
Chicken Cystatin ◽  
Leu 7

The interaction between five N-terminally truncated forms of chicken cystatin (starting at Leu-7, Leu-8, Gly-9, Ala-10 and Asp-15) and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The u.v. absorption, near-u.v. c.d. and fluorescence emission difference spectra for the interactions with papain were all similar to the corresponding spectra for intact cystatin. The second-order association rate constants at 25 degrees C, pH 7.4, I 0.15, for the binding of the truncated forms to papain varied about 2-fold, from 6 x 10(6) to 1.5 x 10(7) M-1.s-1, and were comparable to the value of 9.9 x 10(6) M-1.s-1 for intact cystatin. In contrast, the rate constants for the dissociation of the complexes with papain increased markedly with increasing extent of truncation, from 7.5 x 10(-6)s-1 for Leu7 cystatin (a truncated form of cystatin having Leu-7 as its N-terminal amino acid) to 1.6s-1 for Ala10-cystatin, whereas the dissociation rate constants for the latter form and Asp15-cystatin were similar. Consequently, the binding affinities between the truncated cystatins and papain decreased in an analogous manner, as was also shown for the interaction with actinidin by equilibrium measurements. Studies of the binding of the truncated cystatins to inactivated papains indicated that small substituents on the active-site cysteine of the enzyme can be accommodated in the complex without any loss of affinity when the N-terminal segment of the inhibitor is removed. Taken together, the results suggest that in the N-terminal region of chicken cystatin only residues preceding Ala-10 participate in the interaction with proteinases. Of these residues, Leu-7 and Leu-8 together account for about two-thirds of the unitary free energy of binding contributed by the N-terminal region, the relative importance of the two residues being dependent on the target proteinase. Both Gly-9 and residues N-terminal of Leu-7 further stabilize the interaction but contribute substantially smaller binding energies than do the two leucine residues.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules

2014 ◽  
Vol 70 (5) ◽  
pp. 1311-1320 ◽  
Author(s):  
Antje Schäfer ◽  
Monika Kuhn ◽  
Hermann Schindelin
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Covalent Attachment ◽  
Adenylation Domain ◽  
Active Site Cysteine ◽  
The Status ◽  
Catalytic Cysteine ◽  
Covalently Linked ◽  
First Time

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1–ubiquitin2arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.

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