scholarly journals Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5′-diphosphate and magnesium-adenosine 5′-triphosphate

1977 ◽  
Vol 165 (2) ◽  
pp. 255-262 ◽  
Author(s):  
R N F Thorneley ◽  
A Cornish-Bowden
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Protein A ◽  
Competitive Inhibitor ◽  
Electron Transfer Reaction ◽  
H2 Evolution ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K′i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 × 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.

scholarly journals Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein

1991 ◽  
Vol 279 (1) ◽  
pp. 81-85 ◽  
Author(s):  
K Fisher ◽  
D J Lowe ◽  
R N F Thorneley
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Electron Flux ◽  
High Electron ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Kinetics Of

The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the ‘Fe-protein cycle’ are independent of the level of reduction of the MoFe-protein.

scholarly journals The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation

1984 ◽  
Vol 224 (3) ◽  
pp. 877-886 ◽  
Author(s):  
D J Lowe ◽  
R N Thorneley
Keyword(s):  
Steady State ◽  
Klebsiella Pneumoniae ◽  
Necessary Condition ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Rapid Quench ◽  
Steady State Kinetic ◽  
H2 Formation

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.

scholarly journals Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex

1989 ◽  
Vol 257 (3) ◽  
pp. 789-794 ◽  
Author(s):  
R N F Thorneley ◽  
N H J Bergström ◽  
R R Eady ◽  
D J Lowe
Keyword(s):  
Electron Transfer ◽  
Transfer Reaction ◽  
Competitive Inhibitor ◽  
Electron Transfer Reaction ◽  
Azotobacter Chroococcum ◽  
First Order ◽  
Fe Protein ◽  
Vanadium Nitrogenase ◽  
Electron Transfer Complex ◽  
Kinetics Of

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.

Nonlinear steady-state kinetics of chloramphenicol acetyltransferase

1991 ◽  
Vol 69 (9) ◽  
pp. 630-634
Author(s):  
M. James C. Crabbe ◽  
Derek Goode
Keyword(s):  
Steady State ◽  
Chloramphenicol Acetyltransferase ◽  
Binding Region ◽  
Medium Effects ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of ◽  
State Kinetic

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.Key words: acetyl coenzyme A, chloramphenicol, antibiotics, enzyme kinetics.

scholarly journals Steady-state kinetics of catecholamine transport by chromaffin-granule ‘ghosts’

1974 ◽  
Vol 144 (2) ◽  
pp. 319-325 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Affinity Constant ◽  
Chromaffin Granule ◽  
Affinity Constants ◽  
Steady State Kinetics ◽  
Low Concentrations ◽  
High Concentrations ◽  
Chromaffin Granule Ghosts ◽  
Kinetics Of

Resealed chromaffin-granule ‘ghosts’ were used to study the steady-state kinetics of catecholamine transport. The pump has a high affinity for (-)-noradrenaline, (-)-adrenaline, tyramine and 5-hydroxytryptamine (serotonin), but a lower affinity for (+)-noradrenaline. The measured rates of incorporation do not conform to Michaelis–Menten kinetics, but affinity constants for the former substrates are in the range 8–18μm. Reserpine is a potent inhibitor. Incorporation as a function of ATP concentration also fails to show simple kinetics; the affinity constant for ATP is deduced to be about 3mm at 1mm-MgCl2. Adenylyl (βγ-methylene)diphosphonate is a competitive inhibitor at low concentrations, but inhibits more strongly at high concentrations. The pump has a transition temperature at 29°C and does not seem to be identical with the Mg2+-stimulated adenosine triphosphatase of chromaffin granules.

The Steady State Kinetics of Plasmin and Trypsin Catalysed Hydrolysis of a Number of Tripeptideanilides

1979 ◽  
Author(s):  
U. Christensen ◽  
H-H. Ipsen
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Ph Range ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
State Kinetic

The steady state kinetic parameters of plasmin and trypsin catalysed hydrolysis of Bz-L-Phe-Val-Arg-pNA, L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA in the pH-range 6-9 are presented. Ionization of catalytically essential enzymic groups accounts satisfactorily for the pH-dependencies of the kinetic parameters for plas-rain and trypsin reactions with Bz-L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA. The protonation of the α-amino group of L-Phe-Val-Arg-pNA and D-Phe-Val-Arg-pNA (pK=7.0) show some additional effect. The values of the catalytic constants for plasmin and trypsin reactions with these p-nitroanilides are alike those normally found for specific ester substrates, indicating that the deacylation steps are rate determining.

scholarly journals The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution

1984 ◽  
Vol 224 (3) ◽  
pp. 895-901 ◽  
Author(s):  
D J Lowe ◽  
R N F Thorneley
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rate Constants ◽  
Competitive Inhibitor ◽  
H2 Evolution ◽  
Protein Ratio ◽  
Mofe Protein ◽  
Fe Protein ◽  
Mutual Displacement ◽  
Kinetics Of

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.

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