Slow transacylation of peptidyladenosine allows analysis of the 2'/3'-isomer specificity of peptidyltransferase

• Published in: Biochemistry (Easton) Vol. 24; no. 21; pp. 5776 - 5780
• Main Authors: Taiji, Mutsuo, Yokoyama, Shigeyuki, Miyazawa, Tatsuo
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.10.1985
• Subjects: Acylation; Acyltransferases - metabolism; Adenosine - analogs & derivatives; Adenosine - chemical synthesis; Adenosine - metabolism; Biological and medical sciences; Chromatography, High Pressure Liquid; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Indicators and Reagents; Magnetic Resonance Spectroscopy; Molecular and cellular biology; Molecular genetics; Peptidyl Transferases - metabolism; Phenylalanine-tRNA Ligase - metabolism; Ribosomes - enzymology; Substrate Specificity; Thermus - enzymology; Translation. Translation factors. Protein processing; Translation; Enzyme; Escherichia coli; Substrate specificity; Bacteria; Stereospecificity; Escherichieae; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00342a013

2'-O-(N-acetyl-L-phenylalanyl-L-phenylalanyl)adenosine and 3'-O-(N-acetyl-L-phenylalanyl-L-phenylalanyl)adenosine (Ac-Phe-Phe-Ado) were chemically synthesized, and these two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC) on an ODS column. By this HPLC method, the abundance ratio of the 2'-isomer and 3'-isomer in equilibrium in aqueous solution at pH 7.0 and 0 degrees C was found to be 0.30:0.70, and the equilibration rate was determined as 0.59 +/- 0.04 min-1. Thus, the rate of transacylation between the 2'-isomer and 3'-isomer of peptidyl-tRNA was found to be much slower than that for the two isomers of aminoacyl-tRNA. The HPLC method was used for isomer analysis of the product of the Escherichia coli ribosomal peptidyltransferase reaction. By the use of an isomerizable analogue, 2'(3')-O-L-phenylalanyladenosine (Phe-Ado), as the acceptor of the N-acetyl-L-[3H]phenylalanine (Ac-[3H]Phe) group in the Ac-[3H]Phe-tRNAPhe.poly(U).70S ribosome system, the reaction product was found exclusively to be the 3'-isomer of Ac-[3H]Phe-Phe-Ado. Thus, the slow transacylation of peptidyladenosine allows the analysis of the 2'/3'-isomer specificity of peptidyltransferase.