DETERMINATION OF ENZYME SPECIFICITY IN A COMPLEX MIXTURE OF PEPTIDE-SUBSTRATES BY N-TERMINAL SEQUENCE-ANALYSIS

被引:40
作者
BIRKETT, AJ
SOLER, DF
WOLZ, RL
BOND, JS
WISEMAN, J
BERMAN, J
HARRIS, RB
机构
[1] VIRGINIA COMMONWEALTH UNIV, DEPT BIOCHEM & MOLEC BIOPHYS, BOX 614, RICHMOND, VA 23298 USA
[2] GLAXO RES LABS, RES TRIANGLE PK, NC USA
[3] VIRGINIA POLYTECH INST & STATE UNIV, DEPT BIOCHEM & NUTR, BLACKSBURG, VA 24061 USA
关键词
BOMBARDMENT MASS-SPECTROMETRY; ATRIAL DIPEPTIDYL CARBOXYHYDROLASE; PERFORMANCE LIQUID-CHROMATOGRAPHY; I-CONVERTING ENZYME; FLOW FAST ATOM; ATRIOPEPTIN-II; HYDROLYSIS; MEPRIN;
D O I
10.1016/0003-2697(91)90129-H
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A method has been developed to determine preferred residue substitutions in the P′ position of peptide substrates for proteolytic enzymes. The method has been validated with four different enzymes; the angiotensin I-converting enzyme, atrial dipeptidyl carboxyhydrolase, bacterial dipeptidyl carboxyhydrolase, and meprin A. A mixture of N-acylated potential peptide-substrates for each of the enzymes was prepared in a single synthesis procedure on the same solid-phase synthesis resin. The peptides were identical in all residue positions except the P′ position to be studied, into which numerous amino acid residues were incorporated on a theoretical equimolar basis. After cleavage and extraction of the peptides from the resin, no attempt was made to purify them individually; the exact concentration of each peptide in the mixture was determined by quantitative amino acid analysis. Incubation of an enzyme with its peptide-substrate mixture at [S] ≪ Km yielded peptide hydrolytic products with newly exposed N-termini. The identity and amount of each hydrolysis product was determined by automated N-terminal sequence analysis. One cycle of sequencing revealed preferred amino acid substitutions in the P′1 position, two cycles the P′2 position, and so forth. Comparison of the rates of production of the various products indicates the preferred substitution in that particular P′ position. New information on the substrate specificities of each of the enzymes tested was obtained and it is clear that this approach can be applied to any protease with a defined (or suspected) point of cleavage in a peptide substrate. © 1991.
引用
收藏
页码:137 / 143
页数:7
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