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Updated: Aug 14, 2026

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 18, 2010
[Study of affinity modification kinetics as an approach to demonstrating cooperativity between substrate-recognition
Abstract:
The dependence of the initial rate of the affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by an phenylalanyl-tRNA analog--N-Br-Ac-[14C]Phe-tRNAPhe against reagent concentration was obtained. The curve runs through maximum therefore it is impossible to describe it by the traditional Kitz and Wilson scheme. The experimental results were treated in assumption of dimeric enzyme, cooperativity in the substrate analog binding and its conversion being taken into account. It was shown that such a treatment of kinetic data of the affinity labelling allows to estimate quantitatively the cooperativity arisen between substrate analog centers of the binding and conversion. The data obtained allow to assume that in the case of phenylalanyl-tRNA analog there is a slight negative cooperativity in the reagent binding (thermodynamic cooperativity) but strong negative cooperativity in reagent conversion (kinetic cooperativity). The results obtained testify the high sensitivity of the kinetic approach for elucidation of centers cooperativity.
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