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 Km values, but did cause variable changes in the kcat and kcat/Km values. Ala substitutions at the P1', P1, and P3 sites decreased the kcat/Km values by 400-700-fold. Similar substitutions at the P3', P2, P4, and P5 sites only reduced the kcat/Km 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 non-branched-chain amino acids (e.g., Ala and alpha-aminobutyric acid) are preferred at this site. Only P1' substitution demonstrated any significant change in Km, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
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Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Liang
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012083:Renin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D010455:Peptides; D012083:Renin; D000409:Alanine