Catalysis of slow C-terminal processing reactions by carboxypeptidase H.
A hypothesis was examined that carboxypeptidase H (CpAse H), which is known to catalyse the release of lysine and arginine from the C-terminus of peptides, can also release histidine, tyrosine, and phenylalanine. Synthetic peptides terminating in -His-Lys or -Tyr-Lys were used as model substrates for the enzyme and amino acid analysis was employed to detect release of the terminal amino acids. With N-acetyl-beta-Ala-Asn-Ala-His-Lys and N-acetyl-beta-Ala-Asn-Ala-Tyr-Lys, which correspond to intermediates in the processing of porcine and human beta-endorphin, lysine was removed rapidly and quantitatively but no release of histidine or tyrosine could be detected. To allow more sensitive analysis, radiolabelled substrates were employed and the amounts of the products formed on incubation with CpAse H were determined after separation by ion-exchange chromatography. With 125I-D-Tyr-Ala-His-Lys-Lys as substrate at pH 5.7, very small amounts of D-Tyr-Ala were released; the main product was D-Tyr-Ala-His. At pH 5.0 the release of histidine from 125I-D-Tyr-Ala-His took place 6,000 times more slowly than the release of lysine from 125I-D-Tyr-Ala-Lys. When the tripeptides were incubated at pH 5 with porcine pituitary secretory granules, the lysine was released rapidly but no release of histidine could be detected. The results demonstrate that CpAse H catalyses the release of C-terminal histidine with great difficulty.(ABSTRACT TRUNCATED AT 250 WORDS)
Smyth
D G
DG
National Institute for Medical Research, Mill Hill, London, England.
Maruthainar
K
K
Darby
N J
NJ
Fricker
L D
LD
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000378:metabolism*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism; D001615:beta-Endorphin / Q000235:genetics
D010455:Peptides; D014443:Tyrosine; D006639:Histidine; D001615:beta-Endorphin; D001120:Arginine; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D008239:Lysine