Interaction of difluoro-oxaloacetate with aspartate transaminase

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

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

Biochemical Journal ◽

10.1042/bj3600727 ◽

2001 ◽

Vol 360 (3) ◽

pp. 727-736 ◽

Cited By ~ 9

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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α-Glucosidase and α-Amylase Inhibitory Compounds from three African Medicinal Plants: An Enzyme Inhibition Kinetics Approach

Natural Product Communications ◽

10.1177/1934578x1701200731 ◽

2017 ◽

Vol 12 (7) ◽

pp. 1934578X1701200

Author(s):

Mohammed Auwal Ibrahim ◽

James Dama Habila ◽

Neil Anthony Koorbanally ◽

Md. Shahidul Islam

Keyword(s):

Medicinal Plants ◽

Competitive Inhibitor ◽

Inhibitory Effect ◽

Inhibition Kinetics ◽

Lead Compounds ◽

Inhibitory Compounds ◽

Steady State Kinetic ◽

Enzyme Inhibition Kinetics ◽

State Kinetic ◽

Uncompetitive Inhibitor

The quest to find new lead compounds with anti-diabetic effects via the inhibition of α-glucosidase and α-amylase had led us to conduct bioassay guided isolation of three African medicinal plants which resulted in the identification of bicyclo[2.2.0]hexane-2,3,5-triol (1), 3β- O-acetyl betulinic acid (2) and 2,7-dihydroxy-4 H-1-benzopyran-4-one (3), as the bioactive compounds. The compounds demonstrated a significant (P < 0.05) inhibitory effect on α-glucosidase and α-amylase activities than acarbose. Steady state kinetic analysis revealed that compounds 1 and 2 inhibited both α-amylase and α-glucosidase in non-competitive patterns whilst compound 3 was an uncompetitive inhibitor of α-glucosidase and a non-competitive inhibitor of α-amylase. In conclusion, the study has identified three new active α-glucosidase and α-amylase inhibitory compounds that could have the potential to retard postprandial hyperglycemia.

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Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase

Biochemical Journal ◽

10.1042/bj2860243 ◽

1992 ◽

Vol 286 (1) ◽

pp. 243-251 ◽

Cited By ~ 9

Author(s):

N L Deans ◽

R D Allison ◽

D L Purich

Keyword(s):

Steady State ◽

Thermal Inactivation ◽

Enzyme Reaction ◽

Kinetic Mechanism ◽

Competitive Inhibitor ◽

Product Inhibition ◽

Maximal Velocity ◽

Steady State Kinetic ◽

Tubulin Tyrosine Ligase ◽

State Kinetic

The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.

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