Kinetic Mechanism and Intrinsic Isotope Effects for the Peptidylglycine α-Amidating Enzyme Reaction†

Biochemistry ◽  
1998 ◽  
Vol 37 (22) ◽  
pp. 8244-8252 ◽  
Author(s):  
Wilson A. Francisco ◽  
David J. Merkler ◽  
Ninian J. Blackburn ◽  
Judith P. Klinman
Keyword(s):  
Isotope Effects ◽  
Enzyme Reaction ◽  
Kinetic Mechanism

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

Biochemistry ◽  
1991 ◽  
Vol 30 (23) ◽  
pp. 5755-5763 ◽  
Author(s):  
Paul M. Weiss ◽  
Sandhya R. Gavva ◽  
Ben G. Harris ◽  
Jeffrey L. Urbauer ◽  
W. W. Cleland ◽  
...  
Keyword(s):  
Malic Enzyme ◽  
Isotope Effects ◽  
Enzyme Reaction

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 Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate

2007 ◽  
Vol 282 (46) ◽  
pp. 33484-33493 ◽  
Author(s):  
William C. Cooper ◽  
Yi Jin ◽  
Trevor M. Penning
Keyword(s):  
Conformational Changes ◽  
Isotope Effects ◽  
Transient State ◽  
Kinetic Mechanism ◽  
Energy Diagram ◽  
Single Turnover ◽  
Kinetic Isotope ◽  
Free Energy Diagram ◽  
Burst Kinetics ◽  
Chemical Event

Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3α-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP+ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP+ controlled kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated kcat, whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.

Mutation of Trp93 of MauG to tyrosine causes loss of bound Ca2+ and alters the kinetic mechanism of tryptophan tryptophylquinone cofactor biosynthesis

2013 ◽  
Vol 456 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Sooim Shin ◽  
Manliang Feng ◽  
Victor L. Davidson
Keyword(s):  
Steady State ◽  
Enzyme Reaction ◽  
Kinetic Mechanism ◽  
Reaction Intermediates ◽  
Reaction Phase ◽  
Tryptophan Tryptophylquinone ◽  
Cofactor Biosynthesis ◽  
Processive Enzyme

Mutagenesis of Trp93 of the dihaem enzyme MauG revealed a role for this residue in binding Ca2+ and created an enzyme that exhibits an extraordinarily long pre-steady-state reaction phase during which reaction intermediates of a processive enzyme reaction accumulate.

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