Positional-Scanning Combinatorial Libraries of Fluorescence Resonance Energy Transfer Peptides for Defining Substrate Specificity of the Angiotensin I-Converting Enzyme and Development of Selective C-
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- 作者：Patrí ; cia A. Bersanetti ; Maria Claudina C. Andrade ; Dulce E. Casarini ; Maria A. Juliano ; Aloysius T. Nchinda ; Edward D. Sturrock ; Luiz Juliano ; and Adriana K. Carmona
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：December 21, 2004
- 年：2004
- 卷：43
- 期：50
- 页码：15729 - 15736
- 全文大小：162K
- 年卷期：v.43,no.50(December 21, 2004)
- ISSN：1520-4995
文摘
Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptideswere used for the analyses of the S3 to S1' subsites of the somatic angiotensin I-converting enzyme (ACE).Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to designselective substrates for the C-domain. Initially, we defined the S1 specificity by preparing a library withthe general structure Abz-GXXZXK(Dnp)-OH [Abz = o-aminobenzoic acid, K(Dnp) = N
-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P1 positionhad higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH,and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P1 so we could define the C-domain selectivity of theS1', S2, and S3 subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domainselectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, whichdemonstrated the highest selectivity for the recombinant ACE C-domain (kcat/Km = 36.7 
M-1 s-1) versusthe N-domain (kcat/Km = 0.51 M-1 s-1). The substrate binding of Abz-LFK(Dnp)-OH with testis ACEusing a combination of conformational analysis and molecular docking was examined, and the resultsshed new light on the binding characteristics of the enzyme.
-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P1 positionhad higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH,and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P1 so we could define the C-domain selectivity of theS1', S2, and S3 subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domainselectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, whichdemonstrated the highest selectivity for the recombinant ACE C-domain (kcat/Km = 36.7 M-1 s-1) versusthe N-domain (kcat/Km = 0.51 M-1 s-1). The substrate binding of Abz-LFK(Dnp)-OH with testis ACEusing a combination of conformational analysis and molecular docking was examined, and the resultsshed new light on the binding characteristics of the enzyme.