Molecular and Thermodynamic Mechanisms of the Chloride Dependent Human Angiotensin-I Converting Enzyme (ACE) [Molecular Biophysics]

December 2nd, 2013 by Yates, C. J., Masuyer, G., Schwager, S. L. U., Mohd, A., Sturrock, E. D., Acharya, K. R.

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I (AngI), bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N and C domains which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry (ITC). The X-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of R522. Substrate interactions in the S2 sub-site were shown to affect chloride affinity in the chloride 2 pocket. The E403-K118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond, as well as interactions of larger substrates in the S2 sub-site, moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate selective nature of chloride dependence in ACE and how this varies between the N and C domains.