Structural characterization and enzymatic activity of the recombinant Ala959 to Ser1066 region of human ace

Abstract

Angiotensin-converting enzyme catalyzes the conversion of angiotensin I to the vasoconstrictor angiotensin II and the hydrolysis of bradykinin (BK). Human somatic angiotensin-converting enzyme has two homologous domains (N and C) that share 60% identity. Although these two regions have high homology, the catalytic site of the C-domain exhibits three-fold greater activity than the N-domain in the hydrolysis of angiotensin I in vivo. The present study aimed to obtain the Ala959 to Ser1066 catalytic region of the C-domain of angiotensin-converting enzyme in a structural conformation that resembles its native form. We amplified the 324-bp sequence corresponding to the catalytic site of C-domain of angiotensin-converting enzyme and cloned this sequence into a pET28 vector. The catalytic site of C-domain of angiotensin-converting enzyme peptide was expressed in a bacterial system, and its purification was performed in one step using a His-tag affinity column. Structural analysis by circular dichroism and fluorescence confirmed that the purified protein is correctly folded, and catalytic site of C-domain of angiotensin-converting enzyme possesses enzymatic activity and is inhibited by lisinopril. This peptide can be used to test new inhibitors and C-domain of angiotensin-converting enzyme substrates because this peptide is easy to produce and this has proven efficient link with these molecules.