Pre-Steady-State Kinetic Studies of Protein-Template-Directed Nucleotide Incorporation by the Yeast Rev1 Protein†

Biochemistry ◽  
2007 ◽  
Vol 46 (46) ◽  
pp. 13451-13459 ◽  
Author(s):  
Craig A. Howell ◽  
Satya Prakash ◽  
M. Todd Washington
Keyword(s):  
Steady State ◽  
Kinetic Studies ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals Pre-Steady State Kinetic Studies of the Fidelity of Nucleotide Incorporation by Yeast DNA Polymerase δ

Biochemistry ◽  
2010 ◽  
Vol 49 (34) ◽  
pp. 7344-7350 ◽  
Author(s):  
Lynne M. Dieckman ◽  
Robert E. Johnson ◽  
Satya Prakash ◽  
M. Todd Washington
Keyword(s):  
Steady State ◽  
Dna Polymerase ◽  
Kinetic Studies ◽  
Dna Polymerase Δ ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
State Kinetic

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.

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