Mechanism of tryptophan indole-lyase: insights from pre-steady-state kinetics and substrate and solvent isotope effects

1989 ◽  
Vol 111 (2) ◽  
pp. 727-730 ◽  
Author(s):  
Robert S. Phillips
Keyword(s):  
Steady State ◽  
Isotope Effects ◽  
Steady State Kinetics ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

scholarly journals Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase

1995 ◽  
Vol 309 (1) ◽  
pp. 347-353 ◽  
Author(s):  
S S Tang ◽  
G G Chang
Keyword(s):  
Steady State ◽  
Glutathione Transferase ◽  
Isotope Effects ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Chemical Mechanism ◽  
Octopus Vulgaris ◽  
Pka Value ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction.

scholarly journals Rate and Product Studies with 1-Adamantyl Chlorothioformate under Solvolytic Conditions

2021 ◽  
Vol 22 (14) ◽  
pp. 7394
Author(s):  
Kyoung Ho Park ◽  
Mi Hye Seong ◽  
Jin Burm Kyong ◽  
Dennis N. Kevill
Keyword(s):  
Rate Constants ◽  
Isotope Effects ◽  
Ion Pairs ◽  
Carbonyl Sulfide ◽  
Activation Parameters ◽  
Chloride Ions ◽  
Reaction Channels ◽  
Decomposition Pathway ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

A study was carried out on the solvolysis of 1-adamantyl chlorothioformate (1-AdSCOCl, 1) in hydroxylic solvents. The rate constants of the solvolysis of 1 were well correlated using the Grunwald–Winstein equation in all of the 20 solvents (R = 0.985). The solvolyses of 1 were analyzed as the following two competing reactions: the solvolysis ionization pathway through the intermediate (1-AdSCO)+ (carboxylium ion) stabilized by the loss of chloride ions due to nucleophilic solvation and the solvolysis–decomposition pathway through the intermediate 1-Ad+Cl− ion pairs (carbocation) with the loss of carbonyl sulfide. In addition, the rate constants (kexp) for the solvolysis of 1 were separated into k1-Ad+Cl− and k1-AdSCO+Cl− through a product study and applied to the Grunwald–Winstein equation to obtain the sensitivity (m-value) to change in solvent ionizing power. For binary hydroxylic solvents, the selectivities (S) for the formation of solvolysis products were very similar to those of the 1-adamantyl derivatives discussed previously. The kinetic solvent isotope effects (KSIEs), salt effects and activation parameters for the solvolyses of 1 were also determined. These observations are compared with those previously reported for the solvolyses of 1-adamantyl chloroformate (1-AdOCOCl, 2). The reasons for change in reaction channels are discussed in terms of the gas-phase stabilities of acylium ions calculated using Gaussian 03.

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine

Biochemistry ◽  
1992 ◽  
Vol 31 (28) ◽  
pp. 6585-6591 ◽  
Author(s):  
Grover L. Waldrop ◽  
Joanne L. Turnbull ◽  
Laura E. Parmentier ◽  
M. H. O'Leary ◽  
W. W. Cleland ◽  
...  
Keyword(s):  
Steady State ◽  
Isotope Effects ◽  
Aspartate Transcarbamoylase ◽  
Steady State Kinetics
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