Residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ACE and ACE2) identified by site-directed mutagenesis

Abstract

Angiotensin-converting enzyme (ACE) and its homologue angiotensin-converting enzyme 2 (ACE2) are critical counter-regulatory enzymes of the renin-angiotensin system, and have been implicated in cardiac function, renal disease, diabetes, atherosclerosis and acute lung injury. Both ACE and ACE2 have catalytic activity that is chloride sensitive and is caused by the presence of the CL1 and CL2 chloride-binding sites in ACE and the CL1 site in ACE2. The chloride regulation of activity is also substrate dependent. Site-directed mutagenesis was employed to elucidate which of the CL1 and CL2 site residues are responsible for chloride sensitivity. The CL1 site residues Arg186, Trp279 and Arg489 of testicular ACE and the equivalent ACE2 residues Arg169, Trp271 and Lys481 were found to be critical to chloride sensitivity. Arg522 of testicular ACE was also confirmed to be vital to the chloride regulation mediated by the CL2 site. In addition, Arg514 of ACE2 was identified as a residue critical to substrate selectivity, with the R514Q mutant, relative to the wild-type, possessing a fourfold greater selectivity for the formation of the vasodilator angiotensin-(1-7) from the vasoconstrictor angiotensin II. The enhancement of angiotensin II cleavage by R514Q ACE2 was a result of a 2.5-fold increase in V(max) compared with the wild-type. Inhibition of ACE2 was also found to be chloride sensitive, as for testicular ACE, with residues Arg169 and Arg514 of ACE2 identified as influencing the potency of the ACE2-specific inhibitor MLN-4760. Consequently, important insights into the chloride sensitivity, substrate selectivity and inhibition of testicular ACE and ACE2 were elucidated.

Figure 1

Chloride‐binding sites of tACE (orange) and ACE2 (red): (A) CL1 binding site; (B) CL2 binding site. Residue numbering for tACE is first. The chloride ion is shown in green and is a fixed position relative to both tACE and ACE2 residues. Residues subjected to site‐directed mutagenesis are shown in bold. Cl⁻ coordinating residues are shown in italic. Cl⁻ is unable to be bound by ACE2 at the CL2 site as a result of the side‐chains of Glu398 and Ser511 projecting into this region.

Figure 2

Expression of wild‐type and mutant variants of tACE and ACE2. Aliquots containing 10 μg of total protein obtained from transfected HEK293 cells were separated by SDS‐PAGE (10% polyacrylamide gel). Detection of tACE (A) and ACE2 (B) was visualized by immunoblotting using specific human polyclonal antibodies.

Figure 3

Effect of chloride ion concentration on the activity of wild‐type tACE and ACE2. Activity assays were carried out in 50 mm HEPES buffer, pH 7.4, containing 0–1 m NaCl. (A) Cleavage of angiotensin I by tACE. (B) Cleavage of angiotensin I by ACE2. (C) Cleavage of angiotensin II by ACE2. Generation of product was determined by HPLC. Values are the mean of triplicate determinations ± standard error.

Figure 4

Activity of tACE and ACE2 variants in the absence and presence of NaCl. Activity assays were carried out in 50 mm HEPES buffer, pH 7.4, containing either 0 mm (open bars) or 100/500 mm (filled bars) NaCl. (A) Cleavage of angiotensin I by tACE variants. (B) Cleavage of angiotensin I by ACE2 variants. (C) Cleavage of angiotensin II by ACE2 variants. Generation of product was determined by HPLC. Numbers denote the fold difference between activity recorded at 0 and 100/500 mm NaCl. Values are the mean of triplicate determinations ± standard error. *P < 0.05; **P < 0.005.

Figure 5

IC₅₀ values of captopril with tACE variants (A) and MLN‐4760 with ACE2 variants (B) in the absence and presence of NaCl. IC₅₀ values were determined in 50 mm HEPES buffer, pH 7.4, containing either 0 mm (open bars) or 500 mm (filled bars) NaCl. (A) IC₅₀ values for captopril inhibition of tACE variants. (B) IC₅₀ values for MLN‐4760 inhibition of ACE2 variants. Enzyme activity was recorded over a range of inhibitor concentrations and used to produce dose–response curves from which the IC₅₀ values were elucidated. Numbers denote the fold difference between the IC₅₀ values recorded at 0 and 500 mm NaCl. Values are the mean of triplicate determinations ± standard error. *P < 0.05.