The Enzyme-Inhibitor Dissociation Constants of α-Chymotrypsin and Three Enantiomorphic Pairs of Competitive Inhibitors1

1951 ◽  
Vol 73 (7) ◽  
pp. 3223-3227 ◽  
Author(s):  
H. T. Huang ◽  
Carl Niemann
Keyword(s):  
Enzyme Inhibitor ◽  
Dissociation Constants

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.

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.

scholarly journals Monoamine Oxidase Inhibition by Kavalactones from Kava (Piper Methysticum)

Planta Medica ◽  
2019 ◽  
Vol 85 (14/15) ◽  
pp. 1136-1142
Author(s):  
Denise Prinsloo ◽  
Sandra van Dyk ◽  
Anél Petzer ◽  
Jacobus P. Petzer
Keyword(s):  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Piper Methysticum ◽  
Experimental Conditions ◽  
Mao Inhibitor ◽  
Mao B ◽  
Mao Inhibition ◽  
Mao A ◽  
Ic50 Values

AbstractMonoamine oxidases (MAOs) are key metabolic enzymes for neurotransmitter and dietary amines and are targets for the treatment of neuropsychiatric and neurodegenerative disorders. This study examined the MAO inhibition potential of kavain and other kavalactones from the roots of kava (Piper methysticum), a plant that has been used for its anxiolytic properties. (±)-Kavain was found to be a good potency in vitro inhibitor of human MAO-B with an IC50 of 5.34 µM. (±)-Kavain is a weaker MAO-A inhibitor with an IC50 of 19.0 µM. Under the same experimental conditions, the reference MAO inhibitor, curcumin, displays IC50 values of 5.01 µM and 2.55 µM for the inhibition of MAO-A and MAO-B, respectively. It was further established that (±)-kavain interacts reversibly and competitively with MAO-A and MAO-B with enzyme-inhibitor dissociation constants (Ki) of 7.72 and 5.10 µM, respectively. Curcumin in turn, displays a Ki value of 3.08 µM for the inhibition of MAO-A. Based on these findings, other kavalactones (dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin) were also evaluated as MAO inhibitors in this study. Yangonin proved to be the most potent MAO inhibitor with IC50 values of 1.29 and 0.085 µM for MAO-A and MAO-B, respectively. It may be concluded that some of the central effects (e.g., anxiolytic) of kava may be mediated by MAO inhibition.

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