Slow transacylation of peptidyladenosine allows analysis of the 2'/3'-isomer specificity of peptidyltransferase.
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.
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D010458:Peptidyl Transferases / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology
D007202:Indicators and Reagents; C047810:2'-O-(N-acetylphenylalanylphenylalanine)adenosine; C047811:3'-O-(N-acetylphenylalanylphenylalanine)adenosine; D000217:Acyltransferases; D010458:Peptidyl Transferases; D010652:Phenylalanine-tRNA Ligase; D000241:Adenosine