Transient Kinetics of the Acetylcholinesterase Catalyzed Hydrolysis of N-Methylindoxyl Acetate

1975 ◽  
Vol 53 (3) ◽  
pp. 380-387 ◽  
Author(s):  
M. H. Sadar ◽  
K. J. Laidler
Keyword(s):  
Steady State ◽  
Conformational Change ◽  
Conformational Changes ◽  
Transient Kinetics ◽  
Concerted Mechanism ◽  
Electric Eel ◽  
Fast Transient ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Addition

An experimental study has been made of the kinetics of the hydrolysis of N-methylindoxyl acetate catalyzed by electric-eel acetylcholinesterase, both in the steady state and the pre-steady state. Stopped-flow and temperature-jump experiments revealed a fast transient and a slow one. The fast transient is correlated with the conventional mechanism [Formula: see text]. The slow transient is attributed to conformational changes involving E or EA. Analysis of it revealed two exponential terms of the form e−λt, and the two λ values were obtained over the temperature range 5.0 to 25.0 °C. The results are interpreted in terms of two alternative mechanisms; in one, the enzyme undergoes a conformational change before it adds on the substrate molecule; in the other, the conformational change occurs after the substrate addition. Both mechanisms may be involved, but the results exclude a concerted mechanism in which the conformational change occurs concurrently with the addition of substrate. Kinetic parameters (ΔS≠ and E) are obtained for this conformational change and for the conversion of EA into EA′ + X.

scholarly journals Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum.

1984 ◽  
Vol 98 (2) ◽  
pp. 709-716 ◽  
Author(s):  
P J Van Haastert ◽  
M M Van Lookeren Campagne
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Catalytic Site ◽  
Transient Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Rate Of Activation ◽  
Stimulation Of

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Steady-state and pre-steady-state kinetics of the mitochondrial F(1)F(o) ATPase: is ATP synthase a reversible molecular machine?

2000 ◽  
Vol 203 (1) ◽  
pp. 41-49 ◽  
Author(s):  
A.D. Vinogradov
Keyword(s):  
Steady State ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Gamma Subunit ◽  
Change Mechanism ◽  
Steady State Kinetics ◽  
Energy Dependent ◽  
Variable Substrate ◽  
Kinetics Of ◽  
Hydrolysis Of

H(+)-ATP synthase (F(1)F(o) ATPase) catalyzes the synthesis and/or hydrolysis of ATP, and the reactions are strongly affected by all the substrates (products) in a way clearly distinct from that expected of a simple reversibly operating enzyme. Recent studies have revealed the structure of F(1), which is ideally suited for the alternating binding change mechanism, with a rotating gamma-subunit as the energy-driven coupling device. According to this mechanism ATP, ADP, inorganic phosphate (P(i)) and Mg(2+) participate in the forward and reverse overall reactions exclusively as the substrates and products. However, both F(1) and F(1)F(o) demonstrate non-trivial steady-state and pre-steady-state kinetics as a function of variable substrate (product) concentrations. Several effectors cause unidirectional inhibition or activation of the enzyme. When considered separately, the unidirectional effects of ADP, P(i), Mg(2+) and energy supply on ATP synthesis or hydrolysis may possibly be explained by very complex kinetic schemes; taken together, the results suggest that different conformational states of the enzyme operate in the ATP hydrolase and ATP synthase reactions. A possible mechanism for an energy-dependent switch between the two states of F(1)F(o) ATPase is proposed.

Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside by cellobiase of Trichoderma viride

1977 ◽  
Vol 55 (1) ◽  
pp. 19-26 ◽  
Author(s):  
R. James Maguire
Keyword(s):  
Steady State ◽  
Trichoderma Viride ◽  
Solution Temperature ◽  
Temperature Range ◽  
A Value ◽  
Steady State Kinetics ◽  
Decreasing Ph ◽  
Ph Curve ◽  
Kinetics Of ◽  
Hydrolysis Of

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol–buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 × 10−3 M to 9 × 10−2 M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate – pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol−1 over the temperature range 5–56 °C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol.The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-β-D-glucoside was examined by pre-steady-state methods in which [Formula: see text], and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 s−1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism [Formula: see text]) (ii) a log rate–pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5–6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol−1 at pH 5.7 over the temperature range 10–60 °C.

Presteady-state kinetics of Bacillus 1,3–1,4-β-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate

2001 ◽  
Vol 357 (1) ◽  
pp. 195-202
Author(s):  
Mireia ABEL ◽  
Antoni PLANAS ◽  
Ulla CHRISTENSEN
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Product Formation ◽  
Lag Phase ◽  
Wild Type ◽  
Induced Fit ◽  
Enzyme Substrate ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Presteady State Kinetics

In the present study the first stopped-flow experiments performed on Bacillus 1,3–1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-d-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100s−1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme–substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

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