scholarly journals The initial-rate kinetics of mouse glutathione S-transferase YfYf. Evidence for an allosteric site for ethacrynic acid

1992 ◽  
Vol 286 (3) ◽  
pp. 981-981
Keyword(s):  
Ethacrynic Acid ◽  
Initial Rate ◽  
Glutathione S Transferase ◽  
Allosteric Site ◽  
Rate Kinetics ◽  
Kinetics Of

scholarly journals The initial-rate kinetics of mouse glutathione S-transferase YfYf. Evidence for an allosteric site for ethacrynic acid

1991 ◽  
Vol 275 (3) ◽  
pp. 703-709 ◽  
Author(s):  
M F Phillips ◽  
T J Mantle
Keyword(s):  
Ethacrynic Acid ◽  
Initial Rate ◽  
Competitive Inhibitor ◽  
Activation Mechanism ◽  
Glutathione S Transferase ◽  
Allosteric Site ◽  
Computer Simulation Program ◽  
Rate Kinetics ◽  
A Subunit ◽  
Inhibition Studies

Mouse glutathione S-transferase GST YfYf (an orthologue of GST P or 7-7 in the rat and of GST pi in the human) was found to have a subunit Mr of 24,500 and cross-reacted with anti-(rat GST YfYf). N-Terminal analysis showed a close similarity to the rat, human and bovine orthologues. On isoelectric focusing the native enzyme had a pI of 8.3 and a pI of 7.3 in the presence of urea. Initial-rate studies with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates and inhibition studies with the product of the enzyme-catalysed conjugation of CDNB and GSH, S-(2,4-dinitrophenyl)glutathione, indicated a rapid-equilibrium random mechanism for the enzyme. The diuretic drug ethacrynic acid was found to be simultaneously a competitive inhibitor and an uncompetitive activator of the enzyme (with CDNB as the substrate whose concentration was varied). By using a computer simulation program (EKPLOT) a model was developed that would explain the experimental data. It is proposed that ethacrynic acid can compete with CDNB at the active site but simultaneously bind to an allosteric site on the enzyme, causing an elevation in the Vmax. for the conjugation of CDNB and GSH. The implications of such an activation mechanism for an enzyme potentially conjugating a range of xenobiotic compounds are discussed.

Succinyl Coenzyme A Synthetase of Escherichia coli: Initial Rate Kinetics of Succinyl-CoA Cleavage and Isotope Exchange Studies

1973 ◽  
Vol 51 (1) ◽  
pp. 44-55 ◽  
Author(s):  
Frank J. Moffet ◽  
W. A. Bridger
Keyword(s):  
Isotope Exchange ◽  
Coenzyme A ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Constant Ratio ◽  
Rate Kinetics ◽  
Kinetics Of ◽  
Succinyl Coenzyme A Synthetase

Initial rate kinetic studies of succinyl coenzyme A synthetase of E. coli in the direction of succinyl-CoA cleavage are consistent with the operation of a partially random sequential kinetic mechanism with initial binding of ADP followed by random association of succinyl-CoA and Pi. The mechanism is analogous to that proposed previously for the succinyl-CoA formation reaction, and thus the kinetic mechanism of the overall reversible succinyl-CoA synthetase reaction appears to be symmetrical.Studies of the kinetics of [Formula: see text] isotope exchange at equilibrium show that this partially random sequential kinetic mechanism is not an exclusive pathway. [Formula: see text] isotope exchange rates did not show complete substrate inhibition when CoA or succinate was varied in constant ratio with Pi. However, when CoA or succinate was varied in constant ratio with succinyl-CoA, nearly complete substrate inhibition was observed. These results can be interpreted in terms of a wide variety of minor pathways of substrate binding and product release available to the enzyme under various conditions.

scholarly journals Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXβ reductase (BVR-B)

2000 ◽  
Vol 345 (2) ◽  
pp. 393-399 ◽  
Author(s):  
Orla CUNNINGHAM ◽  
Michael G. GORE ◽  
Timothy J. MANTLE
Keyword(s):  
Fluorescence Quenching ◽  
Ferric Iron ◽  
Initial Rate ◽  
Pyridine Nucleotide ◽  
Flavin Reductase ◽  
Ferric Reductase ◽  
Competitive Kinetics ◽  
Rate Kinetics ◽  
Rapid Equilibrium ◽  
Kinetics Of

The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXβ reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a Kd of 15.8 μM was calculated. Equilibrium fluorescence quenching experiments indicated a Kd of 0.55 μM, suggesting that an enzyme-NADPH encounter complex (Kd 15.8 μM) isomerizes to a more stable ‘nucleotide-induced’ conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with Km values of 52 μM, 125 μM and 53 μM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a Ki of 76 μM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIα, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (Ki 0.59 μM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXβ reductase has also been shown to exhibit ferric reductase activity, with an apparent Km of 2.5 μM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-α isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXβ reductase.

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