scholarly journals A kinetic study of the interaction between mitochondrial F1 adenosine triphosphatase and adenylyl imidodiphosphate and guanylyl imidodiphosphate

1983 ◽  
Vol 210 (3) ◽  
pp. 727-735 ◽  
Author(s):  
F J F Belda ◽  
F G Carmona ◽  
F G Cánovas ◽  
J C Gómez-Fernández ◽  
J A Lozano
Keyword(s):  
Rate Constant ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Reaction Scheme ◽  
F1 Atpase ◽  
Inhibitor Complex ◽  
Change Mechanism ◽  
Mechanism Of Hydrolysis ◽  
Rate Kinetics ◽  
Inhibited Enzyme

1. The presence of 5′-adenylyl imidodiphosphate, a non-hydrolysable analogue of ATP, in the solution used to assay the soluble bovine heart mitochondrial F1-ATPase produced slow competitive inhibition. If the enzyme was preincubated with the inhibitor before the substrate, MgATP, was added, a partial re-activation was obtained. 2. The slow inhibitory process showed first-order rate kinetics, and therefore it seems likely that a conformational change of the enzyme occurs following a faster binding process. A reaction scheme is suggested. At pH 7.8 the rate constant for the inhibition reaction was calculated to be 6.7 × 10(-2)s-1 and that for the re-activation 3.8 × 10(-3)s-1, with Keq. 17.6, indicating that the inhibited enzyme-inhibitor complex will be favoured over the non-inhibited enzyme-inhibitor complex. 3. The presence of 5′-guanylyl imidodiphosphate in the solution used to assay F1-ATPase produced rapid competitive inhibition, which was then slowly reversed until a steady state was reached. This might be explained by a rapid but reversible shift of the inhibition pathway induced by this non-hydrolysable analogue of ATP. A complex rate constant for the displacement of the inhibitor by the substrate of 7.6 × 10(-3)s-1 was calculated. 4. The results are discussed in the light of other recent observations about binding of 5′-adenylyl imidodiphosphate to F1-ATPase and with reference to the binding-site-change mechanism of hydrolysis of ATP by F1-ATPase.

scholarly journals Inactivation of Δ5-3-oxo steroid isomerase with active-site-directed acetylenic steroids

1981 ◽  
Vol 193 (1) ◽  
pp. 217-227 ◽  
Author(s):  
T M Penning ◽  
D F Covey ◽  
P Talalay
Keyword(s):  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Covalent Bond ◽  
Addition Product ◽  
Ring Structure ◽  
Dependent Manner ◽  
Compound I ◽  
Inhibitor Complex ◽  
Acetylenic Ketone

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 × 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 × 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.

scholarly journals The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation

1988 ◽  
Vol 250 (3) ◽  
pp. 827-834 ◽  
Author(s):  
B B Hasinoff ◽  
J P Davey
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Kinetic Studies ◽  
Slow Inactivation ◽  
Radical Species ◽  
Inhibitor Complex ◽  
Antitumour Drug ◽  
Strong Contrast

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.

m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation Complement and Fibrinolytic Serine Proteases

1979 ◽  
Author(s):  
D Bing ◽  
D Robison ◽  
J Andrews ◽  
R Laura
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inhibitor ◽  
Molecular Model ◽  
Factor Xa ◽  
Affinity Labeling ◽  
Catalytic Center ◽  
Inhibitor Complex ◽  
Polypeptide Chains ◽  
Kinetics Of

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F] is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component CIS and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 x 10-3sec-1), the kinetics of inactivation are consistent with inhibition proceding via formation of a specific enzyme-inhibitor complex analogous to a Michaelis-Menton complex (KL - 115-26 μM), and diisopropylfluorophosphate or p-amidino-phenylmethanesulfonyfluoride Prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cationic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants

Optimal Duration of the Preincubation Phase in Enzyme Inhibition Experiments

2020 ◽  
Author(s):  
Petr Kuzmic
Keyword(s):  
Enzyme Inhibition ◽  
Dose Response ◽  
Enzyme Inhibitor ◽  
Single Point ◽  
Association Rate ◽  
Weakly Bound ◽  
Inhibitor Complex ◽  
Dissociation Equilibrium ◽  
Optimal Duration ◽  
Algebraic Formula

This report describes an algebraic formula to calculate the optimal duration of the pre-incubation phase in enzyme-inhibition experiments, based on the assumed range of expected values for the dissociation equilibrium constant of the enzyme–inhibitor complex and for the bimolecular association rate constant. Three typical experimental scenarios are treated, namely, (1) single-point primary screening at relatively high inhibitor concentrations; (2) dose-response secondary screening of relatively weakly bound inhibitors; (3) dose-response screening of tightly-bound inhibitors.

A Very Short Hydrogen Bond Provides Only Moderate Stabilization of an Enzyme-Inhibitor Complex of Citrate Synthase

Biochemistry ◽  
1994 ◽  
Vol 33 (25) ◽  
pp. 7753-7759 ◽  
Author(s):  
Ken C. Usher ◽  
S. James Remington ◽  
David P. Martin ◽  
Dale G. Drueckhammer
Keyword(s):  
Hydrogen Bond ◽  
Citrate Synthase ◽  
Enzyme Inhibitor ◽  
Inhibitor Complex ◽  
Short Hydrogen Bond

scholarly journals Kinetic properties of the primary inhibitor of plasmin from human plasma

1977 ◽  
Vol 163 (2) ◽  
pp. 389-391 ◽  
Author(s):  
U Christensen ◽  
I Clemmensen
Keyword(s):  
Human Plasma ◽  
Enzyme Inhibitor ◽  
Kinetic Properties ◽  
Inhibitor Complex ◽  
Rapid Formation ◽  
Plasmin Inhibitor

The interaction of human plasmin with the newly discovered alpha2-plasmin inhibitor was investigated. It was found from rate measurements that the reaction involves the rapid formation of a first enzyme-inhibitor complex, followed by the slow irreversible transition to another complex. L-Lysine influences the first step, but not the second.

Enzyme-Inhibitor Complex in a Tryptophan-Requiring Mutant of Neurospora crassa

Science ◽  
1957 ◽  
Vol 126 (3282) ◽  
pp. 1068-1069 ◽  
Author(s):  
S. R. SUSKIND ◽  
L. I. KUREK
Keyword(s):  
Neurospora Crassa ◽  
Enzyme Inhibitor ◽  
Inhibitor Complex

scholarly journals Structure of Enzyme-Inhibitor Complex Determined by Neutron Crystallography

2013 ◽  
Vol 55 (1) ◽  
pp. 47-51
Author(s):  
Taro TAMADA ◽  
Motoyasu ADACHI ◽  
Kazuo KURIHARA ◽  
Ryota KUROKI
Keyword(s):  
Enzyme Inhibitor ◽  
Inhibitor Complex ◽  
Neutron Crystallography
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