Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme

Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl -CoA dehalogenase, forming a transient covalent link at the 4-position of t he benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in to tal inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al,). The present work i nvestigates complexes formed by D145N dehalogenase and substrate or product . Time-resolved absorption and Raman difference spectroscopic data show tha t these systems evolve rapidly with time. For the substrate complex, initia lly the absorption and Raman spectra show the signatures of the substrate b ound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resemb ling the un-ionized product in the active site of wild-type dehalogenase. S imilarly, for the product complex, the absorption and Raman spectra initial ly show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the unionized produc t bound to wild-type enzyme. It is proposed that product bound to the activ e site of asparagine 145 dehalogenase catalyzes the deamidation of the aspa ragine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 1 45 in the major enzyme population. Thus, conversions of substrate to produc t and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidati on occurs with a rate constant of 0.0024 h(-1). The rate of product-catalyz ed deamidation in Hepes buffer at 22 degrees C was measured by stopped-flow kinetics to be 0.024 s(-1), 36000 times faster than the spontaneous proces s. A feature near 1570 cm(-1) could be observed in the early Raman spectra of both substrate and product-enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-l ike species formed by the attack of the product's 4-O- group on the carbony l of asparagine's side chain and the subsequent release of ammonia. A react ion scheme is proposed, incorporating these observations.