Active site renin

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

In summary, the results with pepsin extend the renin data reported by Szelke and Boger and strongly support the postulate of Boger that statine is an analog of a dipeptide tetrahedral intermediate.(20) The C-3 hydroxyl group hydrogen bonds to Asp-213 (220) and Asp-33(35) and displaces a "bound" water molecule from the active site. The isobutyl side chain of statine corresponds to the PI substituent that binds to the SI subsite on the enzyme. The C-1 and C-2 atoms of statine serve to span... 

The three-dimensional structures of human (left) and mouse renins (right) showing oligopeptide inhibitors bound in the active site cleft. The cleft lies between the N- and C-terminal domains of the enzyme and is approximately perpendicular to the plane of the page. It can accommodate 9-10 residues with the substrate/inhibitor bound in an extended conformation. The catalytic aspartic acid residues (not shown) are centrally placed at the base of the cleft. 

The active-site cleft has a less open arrangement in renins than in the other aspartic proteinases. Many loops as well as the helix hc (residues 224-236) belonging to the C-domain (residues 190-302) are significantly closer to the active site in the renin structures compared to those of endothiapepsin-inhibitor complexes. This is partly due to a difference in relative position of the rigid body comprising the C-domain. For instance, there is a domain rotation of 4° and translation of 0.1 A in the human renin complex with respect to the endothiapepsin-difluorostatone complex. 

The entrance to the active-site cleft is made even narrower in renins as a consequence of differences in the positions and composition of several well-defined loops and secondary structure elements. Unique to the renins is a cis proline, Prol 11, which caps a helix (hN2) and contributes to the subsites S3 and... 

This rather rigid poly-proline loop, together with the loop comprised of residues 241-250, lies on either side of the active site flap formed by residues 72-81. Hence, in the renins, the cleft is covered by the flaps from both lobes rather than from the N-lobe alone as in other pepsin-like aspartic proteinases. This gives renin a superficial similarity to the dimeric, retroviral proteinases where each subunit provides an equivalent flap that closes down on top of the inhibitor [44,45]. 

The counterregulations induced by the fall in angiotensin II, which stimulates renin release and increases de novo angiotensin I production, which competes with inhibitor at the active sites of ACE (233)... 

It is important to realize how difficult it is to define the in vivo inhibition of ACE. The enzyme may be explored for its N-terminal active sites by measurement of a constant and maximally increased level of N-acetyl SDKP in plasma and urine (211), but the residual activity of the C-ter-minal sites is probably not being measured. The methods for in vitro measurement of plasma ACE, except perhaps that described by Nuss-berger et al. (256), do not appropriately quantify global ACE inhibition. Moreover, the consequences of enzyme blockade are modified by secondary activation of the RAS (233). Residual amounts of angiotensin II secondary to a reactive rise in renin and angiotensin I may explain why the administration of an angiotensin II antagonist still has an additive effect on blood pressure and possibly on the heart, the vessels, and the kidney when added to certain doses of ACE inhibitors (228, 229, 257). 

Figure 9.17. Three Classes of Proteases and Their Active Sites. These examples of a cysteine protease, an aspartyl protease, and a metalloprotease use a histidine-activated cysteine residue, an aspartate-activated water molecule, and a metal-activated water molecule, respectively, as the nucleophile. The two halves of renin are in blue and red to highlight the approximate twofold symmetry of aspartyl proteases.

A unique biochemical target in the HIV-1 replication cycle was revealed when HIV protease was cloned and expressed " in Escherichia coli. HIV protease is an enzyme that cleaves gag-pro propeptides to yield active enzymes that function in the maturation and propagation of new virus. The catalytically active protca.se is a. symmetric dimer of two identical 99 amino acid subunits, each contributing the triad Asp-Thr-Gly to the active site." The homodimer is unlike monomeric asparlyl protea.ses (renin, pepsin, cathep-sin D). which also have different. substrate specificities. The designs of. some inhibitors for HIV-1 protease exploit the C2 symmetry of the enzyme. HIV-1 protease has active site speclnc ity for the triad Tyr-Phe-Pro in the unit Ser-(Thr)-Xaa-Xaa-Tyr-Phc-Pm. whenr Xaa is an arbitrary amino acid. 

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