What is it about?
DTNB inactivates this enzyme by reacting with a single Cys residue in the active-site cleft. This blocks binding of NAD+ and so, conversely, NAD+ protects against inactivation by DTNB. In a previous paper we had analysed this reaction and also protection by coenzyme assuming a simple second order reaction between 'E' and DTNB. In fact this paper shows the inactivation reaction shows saturation kinetics as you increase DTNB concentration, indicating a non-covalent E-DTNB complex prior to covalent reaction. Having analysed this, the paper goes on to show how the reaction can be used to obtain correct dissociation constants for coenzymes or any other protecting ligand.
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Why is it important?
This methodology and analysis are very likely to be applicable to other inactivation processes with different reagents and different enzymes. It iunderlines the importance of tessting ones assumptions.
Read the Original
This page is a summary of: Initial formation of a non-covalent enzyme-reagent complex during the inactivation of clostridial glutamate dehydrogenase by Ellman's reagent: determination of the enzyme's dissociation constant for the binary complex with NAD+ from protection studies, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, December 1994, Elsevier,
DOI: 10.1016/0167-4838(94)90188-0.
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