Full kinetic analysis of a rhodium-catalyzed hydroformylation: beyond the rate-limiting step picture

2015 ◽  
Vol 5 (1) ◽  
pp. 129-133 ◽  
Author(s):  
U. Gellrich ◽  
T. Koslowski ◽  
B. Breit
Keyword(s):  
Steady State ◽  
Kinetic Analysis ◽  
Transition States ◽  
Single Step ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
State Approximation ◽  
Steady State Approximation ◽  
Rate Limiting

A complete dynamic kinetic analysis beyond the steady state approximation of the rhodium-catalyzed hydroformylation with the 6-DPPon ligand is presented. The results show that not one single step but several transition states and intermediates control the selectivity and activity of the catalysis.

scholarly journals Kinetics and mechanism of action of muscle pyruvate kinase

1978 ◽  
Vol 169 (1) ◽  
pp. 39-54 ◽  
Author(s):  
Leighton G. Dann ◽  
Hubert G. Britton
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Pyruvate Kinase ◽  
Alternative Pathway ◽  
Slow Step ◽  
Velocity Data ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Muscle Pyruvate Kinase ◽  
Rate Limiting

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg2+. 2. Methods of preparing [32P]phosphoenolpyruvate from [32P]Pi in high yield and determining [32P]-phosphoenolpyruvate and [8-14C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [32P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-14C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [32P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg2+, with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-14C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-14C]-pyruvate or [γ-32P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.

scholarly journals Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

1990 ◽  
Vol 265 (3) ◽  
pp. 899-902 ◽  
Author(s):  
T R Hawkes ◽  
T Lewis ◽  
J R Coggins ◽  
D M Mousdale ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Lag Phase ◽  
Chorismate Synthase ◽  
Phosphate Release ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state ‘burst’ or ‘lag’ phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

scholarly journals Characterization of a Baculovirus-Encoded RNA 5′-Triphosphatase

1998 ◽  
Vol 72 (9) ◽  
pp. 7057-7063 ◽  
Author(s):  
Christian H. Gross ◽  
Stewart Shuman
Keyword(s):  
Steady State ◽  
Product Formation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Mrna Capping ◽  
Steady State Rate ◽  
Rna Triphosphatase ◽  
State Rate ◽  
Nuclear Polyhedrosis ◽  
Rate Limiting

ABSTRACT Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5′-triphosphatase that hydrolyzes the γ-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 μM γ-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min−1 and converts ATP to ADP with V max of 8 min−1and Km of 25 μM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5′ ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.

Rate-limiting step. Does it exist in the non-steady state?

1997 ◽  
Vol 52 (14) ◽  
pp. 2291-2297 ◽  
Author(s):  
Miloslav Pekař ◽  
Josef Koubek
Keyword(s):  
Steady State ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

scholarly journals Some kinetic and steady-state properties of sodium channels after removal of inactivation.

1981 ◽  
Vol 77 (1) ◽  
pp. 1-22 ◽  
Author(s):  
G S Oxford
Keyword(s):  
Steady State ◽  
Activation Kinetics ◽  
Turn On ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Voltage Dependent ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Secondary Activation ◽  
Pronase Treatment

To study the kinetic and steady-state properties of voltage-dependent sodium conductance activation, squid giant axons were perfused internally with either pronase or N-bromoacetamide and voltage clamped. Parameters of activation, tau m and gNa(V), and deactivation, tau Na, were measured and compared with those obtained from control axons under the assumption that gNa oc m3h of the Hodgkin-Huxley scheme. tau m(V) values obtained from the turn-on of INa agree well with control axons and previous determinations by others. tau Na(V) values derived from Na tail currents were also unchanged by pronase treatment and matched fairly well previously published values. tau m(V) obtained from 3 x tau Na(V) were much larger than tau m(V) obtained from INa turn-on at the same potentials, resulting in a discontinuous distribution. Steady-state In (gNa/gNa max - gNa) vs. voltage was not linear and had a limiting logarithmic slope of 5.3 mV/e-fold gNa. Voltage step procedures that induce a second turn-on of INa during various stages of the deactivation (Na tail current) process reveal quasiexponential activation at early stages that becomes increasingly sigmoid as deactivation progresses. For moderate depolarizations, primary and secondary activation kinetics are superimposable. These data suggest that, although m3 can describe the shape of INa turn-on, it cannot quantitatively account for the kinetics of gNa after repolarization. Kinetic schemes for gNa in which substantial deactivation occurs by a unique pathway between conducting and resting states are shown to be unlikely. It appears that the rate-limiting step in linear kinetic models of activation may be between a terminal conducting state and the adjacent nonconducting intermediate.

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