THIMET OLIGOPEPTIDASE SPECIFICITY - EVIDENCE OF PREFERENTIAL CLEAVAGE NEAR THE C-TERMINUS AND PRODUCT INHIBITION FROM KINETIC-ANALYSIS OF PEPTIDE HYDROLYSIS

被引:24
作者
KNIGHT, CG
DANDO, PM
BARRETT, AJ
机构
[1] Department of Biochemistry, Strangeways Research Laboratory
关键词
D O I
10.1042/bj3080145
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 K-m. 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.
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页码:145 / 150
页数:6
相关论文
共 16 条
[1]   CHARACTERIZATION OF THE BACTERIAL METALLOENDOPEPTIDASE PITRILYSIN BY USE OF A CONTINUOUS FLUORESCENCE ASSAY [J].
ANASTASI, A ;
KNIGHT, CG ;
BARRETT, AJ .
BIOCHEMICAL JOURNAL, 1993, 290 :601-607
[2]  
Atherton E, 1989, SOLID PHASE PEPTIDE
[3]   OLIGOPEPTIDASES, AND THE EMERGENCE OF THE PROLYL OLIGOPEPTIDASE FAMILY [J].
BARRETT, AJ ;
RAWLINGS, ND .
BIOLOGICAL CHEMISTRY HOPPE-SEYLER, 1992, 373 (07) :353-360
[4]  
BARRETT AJ, 1995, IN PRESS METHODS ENZ, V248
[5]   1-HYDROXY-7-AZABENZOTRIAZOLE - AN EFFICIENT PEPTIDE COUPLING ADDITIVE [J].
CARPINO, LA .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1993, 115 (10) :4397-4398
[6]   SOLUBLE METALLOENDOPEPTIDASE FROM RAT-BRAIN - ACTION ON ENKEPHALIN-CONTAINING PEPTIDES AND OTHER BIOACTIVE PEPTIDES [J].
CHU, TG ;
ORLOWSKI, M .
ENDOCRINOLOGY, 1985, 116 (04) :1418-1425
[7]  
CORNISHBROWN A, 1976, PRINCIPLES ENZYME KI, P143
[8]  
COX BG, 1994, MODERN LIQUID PHASE, P27
[9]   HUMAN THIMET OLIGOPEPTIDASE [J].
DANDO, PM ;
BROWN, MA ;
BARRETT, AJ .
BIOCHEMICAL JOURNAL, 1993, 294 :451-457
[10]   A SELECTIVE ASSAY FOR ENDOOLIGOPEPTIDASE A BASED ON THE CLEAVAGE OF FLUOROGENIC SUBSTRATE STRUCTURALLY RELATED TO ENKEPHALIN [J].
JULIANO, L ;
CHAGAS, JR ;
HIRATA, IY ;
CARMONA, E ;
SUCUPIRA, M ;
OLIVEIRA, ES ;
OLIVEIRA, EB ;
CAMARGO, ACM .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1990, 173 (02) :647-652