Determination of Substrate Specificity for Peptide Deformylase through the Screening of a Combinatorial Peptide Library
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- 作者：Yun-Jin Hu ; Yaoming Wei ; Ying Zhou ; P. T. Ravi Rajagopalan ; and Dehua Pei
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：January 12, 1999
- 年：1999
- 卷：38
- 期：2
- 页码：643 - 650
- 全文大小：71K
- 年卷期：v.38,no.2(January 12, 1999)
- ISSN：1520-4995
文摘
Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of theN-terminal formyl group from nascent polypeptides in eubacteria. In vivo, the deformylase is capable ofdeformylating most of the polypeptides in a bacterial cell, which contain diverse N-terminal sequences.In this work, we have developed a combinatorial method to systematically examine the sequence specificityof peptide deformylase. A peptide library that contains all possible N-terminally formylated tetrapeptideswas constructed on TentaGel resin, with a unique peptide sequence on each resin bead. Limited treatmentwith the Escherichia coli deformylase resulted in the deformylation of those peptides that are the mostpotent substrates of the enzyme. By using an enzyme-linked assay, the beads containing the deformylatedpeptides were identified and isolated. Peptide sequence analysis using matrix-assisted laser desorptionionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acidexcept for aspartate and glutamate; Z = lysine, arginine, tyrosine, or phenylalanine), for the E. coli enzyme.The deformylase is also capable of efficient deformylation of formyl-Phe-Tyr-(Phe/Tyr) peptides. Theseresults demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylatesdifferent peptides at drastically different rates. In addition, the selectivity of peptide deformylase for theN-formyl over the N-acetyl group has been studied with N-
-fluoroacetyl peptides, and the results suggestthat both electronic and steric factors are responsible for the observed specificity. The deformylase wasalso shown to exhibit esterase activity. These results will facilitate the design of specific deformylaseinhibitors as potential antibacterial agents. This combinatorial method should be generally applicable tothe study of the substrate specificity of other acylases and peptidases.
-fluoroacetyl peptides, and the results suggestthat both electronic and steric factors are responsible for the observed specificity. The deformylase wasalso shown to exhibit esterase activity. These results will facilitate the design of specific deformylaseinhibitors as potential antibacterial agents. This combinatorial method should be generally applicable tothe study of the substrate specificity of other acylases and peptidases.