Substrate binding properties of converting enzyme using a series of p-nitrophenylalanyl derivatives of angiotensin I.
The binding properties of angiotensin I for the active site of rabbit lung converting enzyme (CE) have been investigated. A series of angiotensin I like substrates, all containing the C-terminal tripeptide, (NO2)Phe-His-Leu, were synthesized by increasing the length of the peptide at the N-terminal end. A total of eight peptides were studied, the largest being [Asn1, (NO2)Phe8]angiotensin I. As determined by thin-layer chromatography, all substrates were hydrolyzed only at the (NO2)Phe-His bond by purified converting enzyme, with the release of the dipeptide, His-Leu. By using an absorbance increase upon hydrolysis, the Michaelis constants and velocity maxima were determined and used to estimate those amino acids in the angiotensin I molecule that contribute significantly to binding to converting enzyme. It was hypothesized that, upon addition or substitution of one or more amino acids to the N-terminal end, a proportional decrease in both KM and Vm is needed in order to conclude that the substrate actually increases its affinity for the enzyme. A test of the proportionality for the variation of KM and Vm was found to be positive for all the substrates, except the N-terminal carbobenzoxy-blocked tripeptide, Z(NO2)Phe-His-Leu. Substitutions near the bond that is hydrolyzed (e.g., proline for the carbobenzoxy group) appear to alter the catalytic properties of CE, while additions far removed from the site of hydrolysis (e.g., the N-terminal tripeptide Asn-Arg-Val) may enhance binding affinity.
D000804:Angiotensin II / Q000031:analogs & derivatives*; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D009574:Nitro Compounds; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D010649:Phenylalanine; D011485:Protein Binding; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D009574:Nitro Compounds; D000804:Angiotensin II; D010649:Phenylalanine; D007703:Peptidyl-Dipeptidase A