The kinetics of inactivation of adenylosuccinate lyase: evidence for a substrate-induced conformational change

1969 ◽  
Vol 47 (6) ◽  
pp. 665-672 ◽  
Author(s):  
W. A. Bridger ◽  
L. H. Cohen
Keyword(s):  
Kinetic Study ◽  
Conformational Change ◽  
Initial Rate ◽  
Alkylating Agents ◽  
Free Enzyme ◽  
The Other ◽  
Adenylosuccinate Lyase ◽  
Product Release ◽  
Rate Kinetics ◽  
Kinetics Of

The kinetics of inactivation of adenylosuccinate lyase by the alkylating agents N-ethyl maleimide and iodoacetamide and by photooxidation have been investigated. The inactivation by the alkylating agents indicates that there are several groups on the enzyme whose reaction affects activity. The presence of AMP, a product of the enzymic reaction, decreases the rate of inactivation by both N-ethyl maleimide and iodoacetamide, while the other product, fumarate, has no effect on the rate. In the case of photoinactivation, the rate is accelerated by the presence of AMP. Fumarate, which again has no effect by itself, causes an overall protection of the enzyme from photoinactivation when added in the presence of AMP. The results, suggesting that fumarate is unable to combine with the free enzyme but does combine with the enzyme–AMP complex, are consistent with the strongly preferred sequence of product release determined by a previous initial-rate kinetic study. Both the kinetics of inactivation and the initial-rate kinetics may be interpreted in terms of a mechanism involving the operation of an enzymic conformational change which is allowed only when either AMP or adenylosuccinate is bound to the enzyme.

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.

Calcium removal kinetics of the sarcoplasmic reticulum ATPase in skeletal muscle

1997 ◽  
Vol 272 (4) ◽  
pp. C1087-C1098 ◽  
Author(s):  
E. E. Burmeister Getz ◽  
S. L. Lehman
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Initial Rate ◽  
Troponin C ◽  
The Other ◽  
Physiological Conditions ◽  
Initial Binding ◽  
Kinetics Of ◽  
Transient And Steady State

The models of the sarcoplasmic reticulum (SR) Ca pump used to simulate Ca kinetics in muscle fibers are simple but inconsistent with data on Ca binding or steady-state uptake. We develop a model of the SR pump that is consistent with data on transient and steady-state Ca removal and has rate constants identified under near-physiological conditions. We also develop models of the other main Ca-binding proteins in skeletal muscle: troponin C and parvalbumin. These models are used to simulate Ca transients in cut fibers during and after depolarizing pulses. Simulations using the full SR pump model are contrasted with simulations using a Michaelis-Menten (MM) approximation to SR pump kinetics. The MM pump underestimates the amount of Ca released during depolarization, underestimates the initial rate of Ca binding by the pump, and overestimates the later rate of Ca pumping. These errors are due to fast initial binding by the SR pump, which is neglected in the MM approximation.

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.

Sign in / Sign up

Export Citation Format

Share Document