SUBSTRATE-SPECIFICITY OF PORCINE RENIN - P1', P1, AND P3 RESIDUES OF RENIN SUBSTRATES ARE CRUCIAL FOR ACTIVITY

Renin, the rate-limiting enzyme in the formation of angiotensin II, is well-known for its stringent substrate specificity. In this study, the biochemical basis for the unusual specificity of renin was investigated by replacing individual amino acids in the octapeptide substrate of renin with Ala. Kinetic analyses of Ala-substituted substrates revealed that the substitutions did not cause significant changes in the K-m values, but did cause variable changes in the k(cat) and k(cat)/K-m values. Ala substitutions at the P1', P1, and P3 sites decreased the k(cat)/K-m values by 400-700-fold. Similar substitutions at the P3', P2, P4, and P5 sites only reduced the k(cat)/K-m values by 2-7-fold. Interestingly, Ala substitution for the P2' Val produced a substrate with an approximately 3-fold increase in activity. These results indicate that the P1', P1, and P3 residues are crucial in determining the substrate specificity of renin. The findings also suggest that the specificity of renin is achieved mainly through substrate discrimination in the transition state, rather than in the ground state. Further studies on the effects of amino acid substitutions at the P2' site revealed that nonbranched-chain amino acids (e.g., Ala and or-aminobutyric acid) are preferred at this site. Only P1' substitution demonstrated any significant change in K,, presumably due to the decreased hydrophobic interactions in the S1' site upon Ala substitution. The species specificity of renin presumably arises from differing P1'-P3' residues in angiotensinogens. For example, the P1'-P3' residues from human and porcine angiotensinogens are Ile-Val-His and Leu-Val-Tyr, respectively. Replacing the P1' Leu with Val in a substrate of porcine renin resulted in a 78-fold decrease in activity, while replacing the P2' Val with Ile had little effect. This result suggests that the P1' residue is more important than the P2' residue in determining the Species specificity of renin.