Dipeptidyl Carboxypeptidase Angiotensin

Angiotensin I converting enzyme is a dipeptidyl carboxypeptidase that cleaves angiotensin I to angiotensin II and the dipeptide histidyl-leucine (His-Leu). The enzyme is bound to the membrane of lung arterial endothelium and is involved in the renin-angiotensin system that regulates blood pressure and fluid balance. 

In the assay reported by Baranowski et al. (1982), the rate of formation of angiotensin II and the dipeptide His-Leu from the substrate angiotensin I was followed. 

The reactant was separated from the product as the fluorescamine derivatives on a reversed-phase HPLC column (Partisil ODS) and eluted isocratically using a two-solvent system. To elute the dipeptide, the solvent was 60% acetonitrile in water diluted 9 1 with 1 M acetic acid, at a final pH of 3.5 (Fig. 9.24). To elute the angiotensin compounds, 38% acetonitrile in 1 M ammonium acetate (pH 4.0) was used (Fig. 9.25). The eluent was monitored with a fluorometer. 

The dipeptidyl carboxypeptidase was prepared from rat lungs. A microsomal fraction was prepared and extracted with detergent (sodium deoxycho-late) and clarified by centrifugation. The supernatant solution was dialyzed against sodium phosphate, and the dialysate was stored frozen. 

In the assay described by Doig and Smiley (1993), the enzyme was assayed by using a synthetic peptide, hippuryl-His-Leu, as substrate and measuring 

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Dipeptidyl Carboxypeptidases.—Dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) has been purified by conventional techniques from a particulate fraction of porcine kidney cortex. The purified enzyme gave two protein bands on standard disc-gel electrophoresis, but showed a single protein component after treatment with neuraminidase. It was concluded that the preparation was a mixture of sialo- and asialo-enzymes and that neuraminic acid residues did not contribute to the catalytic activity. 

ACE is a rather nonspecific peptidase that can cleave C-terminal dipeptides from various peptides (dipeptidyl carboxypeptidase). As kininase 11, it contributes to the inactivation of kinins, such as bradykinin. ACE is also present in blood plasma however, enzyme localized in the luminal side of vascular endothelium is primarily responsible for the formation of angiotensin 11. The lung is rich in ACE, but kidneys, heart, and other organs also contain the enzyme. 

This zinc-dependent enzyme [EC 3.4.15.1] (also known as dipeptidyl carboxypeptidase I, dipeptidyl-dipeptidase A, kininase II, peptidase P, and carboxycathepsin) catalyzes the release of a C-terminal dipeptide at a neutral pH. The enzyme will also act on bradykinin. The presence of prolyl residues in angiotensin I and in bradykinin results in only single dipeptides being released due to the activity of this enzyme, a protein which belongs to the peptidase M2 family. The enzyme is a glycoprotein, generally membrane-bound, that is chloride ion-dependent. 

Ryan, J.W. 1988. Angiotensin-converting enzyme, dipeptidyl carboxypeptidase I, and its inhibitor. 

Metalloproteases include carboxypeptidase [1, 29, 30], ACE (a dipeptidyl carboxypeptidase) [13, 31] and a variety of matrix metalloproteases (matrixins or MMPs) [1, 32, 33]. ACE catalyzes the formation of the vasoconstrictive hormone angiotensin II from angiotensin I, some ACE inhibitors being important anti-hypertensive drugs [13, 31]. 

Dipeptidyl carboxypeptidases remove the C-terminal dipeptide intact and therefore are analogous to the dipeptidyl aminopeptidases such as cathepsin C. One such enzyme, angiotensin-converting enzyme, is important biologically for converting angiotensin I into the hypertensive angiotensin II (see Section 9.3). This enzyme does not hydrolyse bonds of the type X—Pro but will hydrolyse Pro—X bonds. The use of dipeptidyl carboxypeptidases for sequence determination would probably increase if pure enzymes were readily available commercially. 

The prohormone Angiotensin I is then cleaved at the Phe8-His9 peptide bond by the relatively nonspecific ACE (also known as kininase II or dipeptidyl carboxypeptidase). ACE is a membrane-bound glycoprotein, found to be in the epithelial cells of pulmonary capillaries, the urogenital tract, the spleen, and the intestine. Incidentally, by removal of the terminal two amino acids from the carboxyl end of des-Asp-Angiotensin I, ACE can produce seven-membered peptide angiotensin III. The kininase responsible for the breakdown of bradykinin is now known to be identical to ACE. 

Fig. 5.26 Interactions between angiotensin-converting enzyme (a dipeptidyl carboxypeptidase) and a substrate (left) or inhibitors (right). Adapted after Cushman ef a/. ...

In contrast, ACE, also known as kininase II, is a zinc protease that is under minimal physiological control. It is not a rate-limiting step in the generation of angiotensin II and is a relatively nonspecific dipeptidyl carboxypeptidase that requires only a tripeptide sequence as a... 

Angiotensin-converting enzyme (ACE) inhibitors are used for the treatment of high blood pressure, and were designated using the carboxypeptidase structure as a model for Zn " protease action. Captopril is a small, potent, orally available, dipeptidyl inhibitor of ACE. 

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