Add time:07/21/2019         Source:sciencedirect.com

Diethyl pyrocarbonate inactivates enoyl-CoA hydratase (l-3-hydroxyacyl-CoA hydro-lyase, EC 4.2.1.17) with a second-order rate constant of 1.3 M−1 ·s −1. Partial protection is given against inactivation by the substrate analogue acetoacetyl-CoA. The single histidine per enzyme subunit is completely modified at a rate considerably faster than inactivation, and enzymatic activity is not restored by treatment with hydroxylamine. No tyrosine, cysteine or tryptophan residues are modified by diethyl pyrocarbonate. However, out of the 22 amino groups per subunit, 2–5 groups do react with diethyl pyrocarbonate, as shown by difference titration with methyl[1-14C]-acetimidate. Destruction of the N-terminal serine residue by periodate oxidation lowers, but does not abolish enzymic activity. Experiments using 3H-labelled diethyl pyrocarbonate show that the loss of 85% of the original activity is accompanied by the incorporation of approx. three carbethoxy groups. One amino acid residue reacts much faster than the others, and is not essential for activity. Of the next two groups reacting, one is apparently essential for activity. Modification with diethyl pyrocarbonate does not lead to any gross changes in the structure of the enzyme. These experiments taken together show that, in contrast to other hydratases, histidine is not involved in the catalytic mechanism of enoyl-CoA hydratase, and suggest that a single lysine residue is important for activity.

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