Resolution of the uncertainty in the kinetic mechanism for the trans-3-Chloroacrylic acid dehalogenase-catalyzed reaction.

Abstract

trans- and cis-3-Chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively) catalyze the hydrolytic dehalogenation of their respective isomers and represent key steps in the bacterial conversion of 1,3-dichloropropene to acetaldehyde. In prior work, a kinetic mechanism for the CaaD-catalyzed reaction could not be unequivocally determined because (1) the order of product release could not be determined and (2) the fluorescence factor for the enzyme species, E*PQ (where P = bromide and Q = malonate semialdehyde, the two products of the reaction) could not be assigned. The ambiguities in the model have now been resolved by stopped-flow experiments following the reaction using an active site fluorescent probe, αY60W-CaaD and 3-bromopropiolate, previously shown to be a mechanism-based inhibitor of CaaD, coupled with the rate of bromide release in the course of CaaD inactivation. A global fit of the combined datasets provides a complete minimal model for the reaction of αY60W-CaaD and 3-bromoacrylate. In addition, the global fit produces kinetic constants for CaaD inactivation by 3-bromopropiolate and implicates the acyl bromide as the inactivating species. Finally, a comparison of the model with that for cis-CaaD shows that for both enzymes turnover is limited by product release and not chemistry.

DOI: 10.1016/j.abb.2017.05.004

Cite this paper

@article{Huddleston2017ResolutionOT, title={Resolution of the uncertainty in the kinetic mechanism for the trans-3-Chloroacrylic acid dehalogenase-catalyzed reaction.}, author={Jamison P Huddleston and Susan C Wang and K A Johnson and Christian P. Whitman}, journal={Archives of biochemistry and biophysics}, year={2017}, volume={623-624}, pages={9-19} }