Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Biochemistry ◽  
1991 ◽  
Vol 30 (35) ◽  
pp. 8702-8709 ◽  
Author(s):  
William L. Sweet ◽  
John S. Blanchard
Keyword(s):  
Transition State ◽  
Glutathione Reductase ◽  
Human Erythrocyte ◽  
Hydride Transfer ◽  
Chemical Mechanism

scholarly journals The effect of copper on human erythrocyte glutathione reductase

1974 ◽  
Vol 5 (7-8) ◽  
pp. 649-653 ◽  
Author(s):  
J.P. Flikweert ◽  
R.K.J. Hoorn ◽  
G.E.J. Staal
Keyword(s):  
Glutathione Reductase ◽  
Human Erythrocyte ◽  
Effect Of Copper

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

scholarly journals Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36

2016 ◽  
Vol 113 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
Myles B. Poulin ◽  
Jessica L. Schneck ◽  
Rosalie E. Matico ◽  
Patrick J. McDevitt ◽  
Michael J. Huddleston ◽  
...  
Keyword(s):  
Transition State ◽  
Isotope Effects ◽  
Histone H3 ◽  
Kinetic Isotope Effects ◽  
Chemical Mechanism ◽  
State Structure ◽  
Transition State Structure ◽  
Chemical Modeling ◽  
Kinetic Isotope ◽  
Human Multiple Myeloma

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf–Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-14C and 36S and secondary Me-3H3, Me-2H3, 5′-14C, and 5′-3H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

scholarly journals Structure and function of yeast alcohol dehydrogenase

2000 ◽  
Vol 65 (4) ◽  
pp. 207-227 ◽  
Author(s):  
Svetlana Trivic ◽  
Vladimir Leskovac
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Primary Structure ◽  
Active Site ◽  
Hydride Transfer ◽  
Kinetic Mechanism ◽  
Structure And Function ◽  
Chemical Mechanism ◽  
And Function ◽  
Font Color

1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer <br><br><font color="red"><b> This article has been corrected. Link to the correction <u><a href="http://dx.doi.org/10.2298/JSC0008609E">10.2298/JSC0008609E</a><u></b></font>

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