Porcine Chymotrypsin A-π, a More Acidic Chymotrypsin

1975 ◽  
Vol 53 (10) ◽  
pp. 1101-1105 ◽  
Author(s):  
Eveline de Médicis ◽  
Louise Bergeron
Keyword(s):  
Steady State ◽  
Kinetic Study ◽  
Competitive Inhibitor ◽  
Catalytic Process ◽  
Binding Conformation ◽  
Bovine Trypsin ◽  
Chymotrypsin B ◽  
Chymotrypsin A ◽  
Ph Profiles

A kinetic study of porcine chymotrypsin A-π revealed two characteristic properties of this type of chymotrypsin:1. Porcine chymotrypsin A-π, like bovine chymotrypsin B-π, does not bind proflavin. which is a competitive inhibitor of bovine trypsin and chymotrypsin A-α.2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low.

scholarly journals Interaction of difluoro-oxaloacetate with aspartate transaminase

1977 ◽  
Vol 161 (2) ◽  
pp. 383-387 ◽  
Author(s):  
P A Briley ◽  
R Eisenthal ◽  
R Harrison ◽  
G D Smith
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Aspartate Transaminase ◽  
Steady State Kinetic ◽  
High Concentrations ◽  
State Kinetic ◽  
Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

KINETIC EQUATIONS FOR ENZYME REACTIONS IN THE PRESENCE OF POWERFUL INHIBITORS

1959 ◽  
Vol 37 (8) ◽  
pp. 1268-1271 ◽  
Author(s):  
Richard M. Krupka ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Kinetic Equations ◽  
Competitive Inhibitor ◽  
Graphical Analysis ◽  
Rate Data ◽  
State Equations ◽  
Enzyme Reactions ◽  
Simple Addition

Steady-state equations are worked out for the case of a competitive inhibitor that is present in concentrations comparable with that of the enzyme; allowance is made for the inhibitor attached to the enzyme. Two cases are considered: in case 1 the enzyme and inhibitor form a simple addition complex, while in case 2 a molecule is split off. Methods of graphical analysis of rate data are described.

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

1989 ◽  
Vol 994 (1) ◽  
pp. 59-63 ◽  
Author(s):  
Patricia J. Harvey ◽  
John M. Palmer ◽  
Hans E. Schoemaker ◽  
Henk L. Dekker ◽  
Ron Wever
Keyword(s):  
Steady State ◽  
Kinetic Study ◽  
Compound I ◽  
Steady State Kinetic ◽  
State Kinetic

A KINETIC STUDY OF THE DEAMINATION OF SOME ADENOSINE ANALOGUES: SUBSTRATE SPECIFICITY OF ADENOSINE DEAMINASE

1966 ◽  
Vol 44 (3) ◽  
pp. 331-337 ◽  
Author(s):  
J. Lyndal York ◽  
G. A. LePage
Keyword(s):  
Substrate Specificity ◽  
Kinetic Study ◽  
Adenosine Deaminase ◽  
Competitive Inhibitor ◽  
Kinetic Constants ◽  
Glycosidic Linkage ◽  
Adenosine Analogues ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Marked Dependence

The kinetic constants Km and Vmax were determined for the deamination by adenosine deaminase of a series of analogues of adenosine containing "fraudulent" sugars. The configuration of the 2′-hydroxyl was found to be important for the binding of enzyme and substrate. The largest effect of changes in sugar structure was on the rate of breakdown of the enzyme–substrate complex to form products, i.e. Vmax. The nature of the configuration in the 3′-position was not important if the 2′-hydroxyl was trans to the glycosidic linkage; however, if the steric arrangement of the 2′-hydroxyl was cis to the glycosidic linkage, then Vmax showed a marked dependence on the nature of the 3′-substituent and its configuration. For instance, Vmax values were for arabinosyl adenine < 3′-deoxyarabinosyl adenine <lyxosyl adenine. 6-N-methyladenosine was found to be a competitive inhibitor of adenosine deaminase, with a Ki of 2 × 10−6M.

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