Thimet oligopeptidase specificity: evidence of preferential cleavage near the C-terminus and product inhibition from kinetic analysis of peptide hydrolysis.
The substrate-size specificity of human thimet oligopeptidase (EC 3.4.24.15) was investigated with oligomers of glycyl-prolyl-leucine (GPL)n where n = 2, 3, 4 and 5. These peptides were cleaved only at Leu-Gly bonds to give GPL as the single final product. Hydrolysis was most rapid with (GPL)3 and slowest with (GPL)5. The more water-soluble oligomers of Gly-Hyp-Leu showed the same trend. (Gly-Hyp-Leu)6 was not hydrolysed, consistent with the previous finding that substrates larger than 17 amino acids are not cleaved by thimet oligopeptidase. The cleavage of (GPL)3 to GPL fitted a sequential first-order model. First-order kinetics were unexpected as the initial substrate concentration was greater than Km. The anomaly was also seen during the cleavage of bradykinin and neurotensin, and in these cases first-order behaviour was due to potent competitive inhibition by the C-terminal product. The sequential mechanism for (GPL)3 breakdown by thimet oligopeptidase does not discriminate between initial cleavages towards the N- or C-terminus. As isoleucine is an unfavourable residue in P1, substrates were made in which selected leucine residues were replaced by isoleucine. GPL--GPI--GPL (where--represents the bond between the tripeptide units) was resistant to hydrolysis and GPI--GPL--GPL was cleaved only at the -Leu-Gly- bond. Experiments with isoleucine-containing analogues of (Gly-Hyp-Leu)4 showed that thimet oligopeptidase preferred to cleave these peptides near the C-terminus.
Knight
C G
CG
Department of Biochemistry, Strangeways Research Laboratory, Worts Causeway, Cambridge, U.K.
Dando
P M
PM
Barrett
A J
AJ
D000595:Amino Acid Sequence; D001920:Bradykinin / Q000737:chemistry; D003094:Collagen / Q000737:chemistry; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010446:Peptide Fragments; D009496:Neurotensin; D003094:Collagen; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D001920:Bradykinin