Molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-I-converting enzyme (ACE)

Abstract

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, 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. 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 Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) 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 subsite 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.

Keywords: Angiotensin-1-converting Enzyme; Biophysics; Chloride Ion Activation; Crystallography; Enzymology; Hypertension; Protein Structure; Thermodynamics.

FIGURE 1.

Overall structure of C-domain ACE mutants. A, location of mutated residues Glu⁴⁰³, Asp⁴⁶⁵, and Arg⁵²² (magenta) in C-domain ACE (cyan; Protein Data Bank code 1O8A (6)), with chloride and zinc ions shown as spheres (green and gray, respectively). B, superposition of C-domain ACE (cyan; Protein Data Bank code 1O8A (6)) with the mutant structures E403R (light pink), D465T (pink), R522K (violet), and R522Q (purple).

FIGURE 2.

Electron density map of mutated residues in C-domain ACE. A weighted difference map was calculated with REFMAC5 and is displayed at 1σ level for E403R (A), D465T (B), R522K + captopril (C), R522K (D), and R522Q (E).

FIGURE 3.

Structural changes from mutations in C-domain ACE. Comparison of local structural changes from mutations in C-domain ACE. Shown are native (cyan) and mutant (magenta) structures where E403R (A), D465T (B), R522K + captopril (C), R522K (D), and R522Q (E) are represented. Chloride and zinc ions are shown as spheres (green and gray, respectively), and water molecules are shown in red. Possible hydrogen bonds are shown as dashed lines.

FIGURE 4.

Effect of chloride concentration on thermodynamic parameters associated with ACE hydrolysis. Shown is the thermodynamic signature for the hydrolysis of HHL, Z-FHL, and angiotensin I at 0 and 20 mm NaCl by the C-domain (A) and N-domain (B). The values for the C-domain with HHL at 300 mm NaCl are included because this is the concentration of maximal activity and chloride saturation for this substrate and domain (whereas 20 mm is the maximum for the other values). C, the ΔΔG, ΔΔH, and −TΔΔS values for the C-domain and N-domain represent the difference in ΔG, ΔH, and −TΔS between 0 and 20 mm (between 0 and 300 mm for HHL with the C-domain). D, relative K_d(app) values (left y axis) and -fold increase in k_cat/K_m from 0 mm to maximal activity (right y axis) for C- and N-domains with HHL, Z-FHL, and AngI. Error bars, S.E.

FIGURE 5.

Kinetic and thermodynamic comparisons for R186H. The k_cat/K_m for the C-domain (black bars) and R186H (white bars) taken as the percentage of the k_cat/K_m for C-domain at 0 mm (A) and 20 mm (B) NaCl. C, the ΔΔG, ΔΔH, and −TΔΔS values for C-domain and R186H represent the difference in ΔG, ΔH, and −TΔS between 0 and 20 mm NaCl.

FIGURE 6.

Trends in chloride binding and activity for R522Q and R522K. Shown is a graphical representation of chloride binding and kinetic values obtained for C-domain (black bars), R522K (gray bars), and R522Q (white bars) with HHL, Z-FHL, and AngI as substrates. A, the K_d(app) values (mm) for chloride binding. *, AngI values were estimated from the report of Liu et al. (35). The k_cat/K_m value was taken as the percentage of the k_cat/K_m for the C-domain at 0 mm (B) and 20 mm (C) NaCl. D, change in thermodynamic parameters for R522Q and R522K. The ΔΔG, ΔΔH, and −TΔΔS values for C-domain, R522Q, and R522K with HHL, Z-FHL, and AngI are shown and represent the difference in ΔG, ΔH, and −TΔS between 0 and 20 mm NaCl.

FIGURE 7.

Model of angiotensin I binding to C-domain ACE. The structure of the C-domain ACE·Ang complex (Protein Data Bank code 4APH (36)) was used to model Angiotensin I before catalysis. The AngI peptide is represented in gray, chloride is shown in green, zinc ion is shown in gray, and water molecules are shown in red. The potential hydrogen bonds are shown in dashed lines. Residue 522 is presented in a stick representation.

FIGURE 8.

Trends in chloride binding and activity for E403R. Shown is a graphical representation of k_cat/K_m kinetic values obtained for the C-domain (black bars) and E403R (white bars) with HHL, Z-FHL, and AngI as substrates. The k_cat/K_m value was taken as the percentage of the k_cat/K_m for the C-domain at 0 mm (A) and 20 mm (B) NaCl. C, change in thermodynamic parameters for E403R. The ΔΔG, ΔΔH, and −TΔΔS values for the C-domain and E403R are shown and represent the difference in ΔG, ΔH, and −TΔS between 0 and 20 mm NaCl (0 and 300 mm for HHL with C-domain).

FIGURE 9.

Comparison of C-domain ACE bound to peptide inhibitor BPPb and the E403R mutant structures. The structures of the C-domain ACE (cyan; Protein Data Bank code 4APJ (36)) and C-domain ACE·BPPb complex (blue; Protein Data Bank code 4APJ (36)) were superposed onto the C-domain E403R structure (magenta). The BPPb peptide is represented in yellow, and the zinc ion is shown in gray (not present in the BPPb complex structure). Interactions with position 403 are shown as dashed lines (colors correspond to the respective structures).

FIGURE 10.

Schematic representation of substrate binding modes in C-domain ACE. Binding of substrate to the active site is shown using an adapted Schechter and Berger diagram (42), with active site subpockets (S2, S1, S1′, and S2′) shown as linked half-spheres and peptides shown as labeled black spheres (P2, P1, P1′, and P2′, with P3+ indicating residues P3–P10 for AngI). Positions of C-domain ACE residues (light blue) are shown above the relevant subsites, with helix α23 containing Arg⁵²², Tyr⁵²³, and Tyr⁵²⁰ shown as a green triangle. Chloride ions (numbered green spheres) are shown, with chloride coordination to Arg⁵²² indicated by a dashed green line and the influence of chloride on Lys⁵¹¹ shown by a green arrow. The active site zinc (gray sphere) indicates the position of the catalytic mechanism between S1 and S1′, with yellow lightning showing the position of the scissile bond for each peptide. Interactions that increase chloride affinity (green-shaded semicircles), those that do not increase chloride affinity (red-shaded semicircles), and interactions that may affect chloride affinity (orange-shaded semicircles) are indicated above each peptide residue.