Hu Y J, Wei Y, Zhou Y, Rajagopalan P T, Pei D
Department of Chemistry, Ohio State Biochemistry Program, The Ohio State University, Columbus 43210, USA.
Biochemistry. 1999 Jan 12;38(2):643-50. doi: 10.1021/bi9820412.
Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of the N-terminal formyl group from nascent polypeptides in eubacteria. In vivo, the deformylase is capable of deformylating 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 specificity of peptide deformylase. A peptide library that contains all possible N-terminally formylated tetrapeptides was constructed on TentaGel resin, with a unique peptide sequence on each resin bead. Limited treatment with the Escherichia coli deformylase resulted in the deformylation of those peptides that are the most potent substrates of the enzyme. By using an enzyme-linked assay, the beads containing the deformylated peptides were identified and isolated. Peptide sequence analysis using matrix-assisted laser desorption ionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acid except 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. These results demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylates different peptides at drastically different rates. In addition, the selectivity of peptide deformylase for the N-formyl over the N-acetyl group has been studied with N-alpha-fluoroacetyl peptides, and the results suggest that both electronic and steric factors are responsible for the observed specificity. The deformylase was also shown to exhibit esterase activity. These results will facilitate the design of specific deformylase inhibitors as potential antibacterial agents. This combinatorial method should be generally applicable to the study of the substrate specificity of other acylases and peptidases.
肽脱甲酰基酶是一种必需的Fe2+金属酶，可催化真细菌中新生多肽N端甲酰基的去除。在体内，脱甲酰基酶能够使细菌细胞中大多数含有不同N端序列的多肽脱甲酰基。在这项工作中，我们开发了一种组合方法来系统地研究肽脱甲酰基酶的序列特异性。在TentaGel树脂上构建了一个包含所有可能的N端甲酰化四肽的肽库，每个树脂珠上有一个独特的肽序列。用大肠杆菌脱甲酰基酶进行有限处理，导致那些作为该酶最有效底物的肽脱甲酰基。通过酶联测定，鉴定并分离出含有脱甲酰基化肽的珠子。使用基质辅助激光解吸电离质谱进行肽序列分析，揭示了大肠杆菌酶的共有序列：甲酰基-甲硫氨酸-X-Z-酪氨酸（X = 除天冬氨酸和谷氨酸外的任何氨基酸；Z = 赖氨酸、精氨酸、酪氨酸或苯丙氨酸）。脱甲酰基酶也能够高效地使甲酰基-苯丙氨酸-酪氨酸-（苯丙氨酸/酪氨酸）肽脱甲酰基。这些结果表明，尽管肽脱甲酰基酶是一种具有广泛特异性的酶，但它使不同肽脱甲酰基的速率差异很大。此外，用N-α-氟乙酰肽研究了肽脱甲酰基酶对N-甲酰基和N-乙酰基的选择性，结果表明电子和空间因素都对观察到的特异性起作用。还显示脱甲酰基酶具有酯酶活性。这些结果将有助于设计作为潜在抗菌剂的特异性脱甲酰基酶抑制剂。这种组合方法通常应适用于研究其他酰基转移酶和肽酶的底物特异性。
