The Dependence of the Enzyme-Inhibitor Dissociation Constants of Several Bifunctional Anionic Competitive Inhibitors of α-Chymotrypsin upon Added Potassium Phosphate1

1957 ◽  
Vol 79 (15) ◽  
pp. 4091-4096 ◽  
Author(s):  
Richard A. Bernhard ◽  
Carl Niemann
Keyword(s):  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Competitive Inhibitors

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 Mechanistic Studies of the Inactivation of TEM-1 and P99 by NXL104, a Novel Non-β-Lactam β-Lactamase Inhibitor

2010 ◽  
Vol 54 (12) ◽  
pp. 5132-5138 ◽  
Author(s):  
Thérèse Stachyra ◽  
Marie-Claude Péchereau ◽  
Jean-Michel Bruneau ◽  
Monique Claudon ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Mechanistic Studies ◽  
Ionization Time ◽  
Inhibitor Complex ◽  
Class A ◽  
Flight Mass Spectrometry ◽  
Covalent Adduct ◽  
Improved Stability

ABSTRACT NXL104 is a potent inhibitor of class A and C serine β-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 β-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 β-lactamases were determined, including partition ratios, dissociation constants (K), rate constants for deactivation (k 2), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies (k 2/K of 3.7 × 105 M−1 s−1 for TEM-1 and 1 × 104 M−1 s−1 for P99) and slow decarbamylation. Complete loss of β-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k 3 value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC50s) were evaluated on selected β-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C β-lactamases. IC50s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the β-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.

scholarly journals A Competitive Inhibitor of Tyrosinase

1951 ◽  
Vol 4 (4) ◽  
pp. 554 ◽  
Author(s):  
C Warner
Keyword(s):  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Competitive Inhibitor ◽  
Enzyme Preparations ◽  
Enzyme Substrate ◽  
Kinetics Of

The kinetics of the activation of catechol by tyrosinase prepared from the potato and the mushroom, and of its inhibition by sodium m-hydroxybenzoate, have been studied. The enzyme-substrate dissociation constants differed markedly between the two enzyme sources (K. potato = 5.OmM, Kg mushroom = O.28mM), as did also the enzyme-inhibitor dissociation constants (K; potato = 2.5mM, Ki mushroom = O.BmM). For both enzyme preparations sodium m-hydroxybenzoate met the requirements of a competitive inhibitor.

Active Site-Dependent Substrate Recognition Plays a Primary Role in Determining the Affinity of the Thrombin-Thrombomodulin Complex for Protein C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 125-125
Author(s):  
Genmin Lu ◽  
Sotheavy Chhum ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Protein C ◽  
Dissociation Constants ◽  
Substrate Recognition ◽  
Primary Role ◽  
Competitive Inhibitors ◽  
Extended Surfaces ◽  
Equilibrium Dissociation Constants ◽  
Anticoagulant Function ◽  
Equilibrium Dissociation

Abstract The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C. The TM-dependent enhancement of the anticoagulant function of IIa has alternately been considered to primarily arise from an allosteric modulation of the active site of the protease or from the ability of TM to bind protein C and participate in its presentation to the enzyme. We have applied established kinetic strategies to investigate the relative contributions of active site interactions versus interactions with extended surfaces in the action of the IIa-TM complex on human protein C. Initial velocity studies yielded equivalent kinetic constants for the hydrolysis of L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroanilide (S2366) by IIa alone or IIa saturated with TM. The reversible active site-directed ligands Nα-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581) and 4-amino-phenylpyruvic acid (APPA) were found to act as classical competitive inhibitors of S2366 cleavage by either IIa or IIa-TM. This finding is consistent with the limited interactions expected between S2366 and the active site of either IIa or IIa-TM. Equivalent inhibition constants were obtained for IIa or IIa-TM with either inhibitor that were in good agreement with directly determined equilibrium dissociation constants for I-2581 (IIa: Kd =25 ± 1 nM, IIa-TM: Kd=16 ± 1 nM) or APPA (IIa: Kd = 530 ± 90 nM, IIa-TM: Kd = 370 ± 40 nM). Similar kinetic and equilibrium dissociation constants obtained for IIa and IIa-TM indicate that obvious changes in the active site of IIa induced by TM are not detectable by this series of active-site directed probes. In contrast to peptidyl substrate cleavage, protein C activation by IIa (Km=177 ± 31 μM, Vmax/Et=0.004±0.001 s−1) was greatly enhanced by the addition of saturating concentrations of TM to form the IIa-TM complex (Km=12 ± 1 μM, Vmax/Et=0.28±0.01 s−1). However, both active site-directed reversible inhibitors continued to act as classical competitive inhibitors of protein C activation by IIa-TM (I-2581: Ki = 20 ± 1 nM; APPA: Ki = 470 ± 20 nM). Mutually exclusive binding interactions of protein C and active site-directed inhibitors indicate a principal role for active site-dependent interactions in determining the affinity of IIa-TM for protein C. This conclusion was further investigated by using a recombinant derivative of protein C with the P1 Arg mutated to Gln (PCR169Q) that rendered the zymogen uncleavable by either IIa or the IIa-TM complex. PCR169Q was a poor inhibitor (Ki>>50 μM) of protein C activation or S2236 cleavage implying a greatly reduced affinity for IIa-TM. These data suggest a primary role for active site-dependent substrate recognition in driving the affinity of IIa-TM for protein C. In contrast to the established role for exosites in action of IIa on many of its other substrates, possible interactions between protein C and extended surfaces within the IIa-TM complex likely contribute in a secondary way in determining the affinity of this enzyme complex for its biological substrate.

Interaction of lactate dehydrogenase isoenzymes with ligands

1988 ◽  
Vol 53 (8) ◽  
pp. 1857-1861 ◽  
Author(s):  
Jana Barthová ◽  
Jana Kučerová ◽  
Sylva Leblová
Keyword(s):  
Lactate Dehydrogenase ◽  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Competitive Inhibitor ◽  
Kinetic Measurements ◽  
Cibacron Blue ◽  
Michaelis Constants ◽  
Sepharose 4B ◽  
Lactate Dehydrogenase Isoenzymes

Isoenzymes of bovine lactate dehydrogenase (H4, H3M, and H2M2) were prepared by affinity chromatography on a 5'-AMP-Sepharose 4B column. The interaction of isoenzymes with two ligands, coenzyme NADH and the competitive inhibitor Cibacron Blue F3GA was followed by means of kinetic measurements and by affinity electrophoresis. The Michaelis constants of NADH were compared with the inhibition constants of Cibacron Blue and dissociation constants of enzyme-inhibitor complexes. It was found that the M subunit of lactate dehydrogenase exhibits always higher affinity both to NADH and Cibacron Blue in comparison to the H subunit. This finding corresponds to the physiological role of lactate dehydrogenase isoenzymes.

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