The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70S ribosomes was investigated in the fMet-tRNA.A-U-G.70S ribosome and AcPhe-tRNA.poly(U).70S ribosome systems by using a series of 2'- and 3'-aminoacyldinucleoside phosphates as acceptors. These chemically synthesized compounds are analogues of the 3' termini of either 2'(3')-, 2'-, or 3'-aminoacyl transfer ribonucleic acids (AA-tRNAs) of the types C-A-aa, C-2'-dA-aa, C-3'-dA-aa, C-3'-dA-3'-NH-aa, and C-2'-dA-2'-NH-aa (aa = Phe, D-Phe, Lys, Leu, Ala, Glu, Pro, Gly, Asp, Met, and alpha-aminoisobutyryl). It was found that the 3'-aminoacyl derivatives of optically active amino acids are much better acceptors of N-formyl-L-methionine (fMet) or N-acetyl-L-phenylalanine (AcPhe) residues than the isomeric 2'-aminoacyl derivatives with affinity constant ratios (KM 2'/3') greater than 100. Likewise, C-A(D-Phe) is a weaker acceptor than the corresponding L derivative C-A-Phe. In contrast, all glycyl derivatives (C-2'-dA-Gly, C-3'-dA-Gly, C-3'-dA-3'-NH-Gly and C-2'-dA-2'-NH-Gly) are good acceptors of the fMet residue, with ratios (KM 2'/3') of approximately 2. On the basis of these results, a model for the stereochemical control of the peptidyl-transferase reaction is proposed. It assigns a major role to the orientation of the amino acid side chain in 2'- or 3'-AA-tRNA. A detailed model of the interaction of the acceptor terminus of 3'-AA-tRNA with the acceptor site of peptidyltransferase is also proposed. The model is strikingly similar to those for the active sites of proteolytic enzymes.

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These measurements were partly supported by the Biomedical Research Support Grant SO-7-RR-05529 from the National Institutes of Health. Assistance of R. W. Schubring during the synthesis of acid 2b is also appreciated. Our thanks are likewise due to Drs.

The chemical syntheses of 2'(3')-O-(L-3-amino-3-phenylpropionyl)adenosine (2e), the corresponding D stereoisomer 2f, 2'(3')-O-(DL-phenylglycyl)adenosine (2g), 2'(3')-O-(N-benzylglycyl)adenosine (2h), and 9(2-O-L-phenylalanyl-beta-D-xylofuranosyl)adenine (3b) are described. Compounds 2e-h were obtained by acylation of 5'-O-(4-methoxytrityl)adenosine with the appropriate N-benzyloxycarbonyl or N-tert-butoxycarbonyl amino acids with dicyclohexylcarbodiimide in pyridine. The corresponding reaction of N-(benzyloxycarbonyl)-D-phenylglycine led to an almost complete racemization of the aminoacyl residue (compounds 2c and 2g). Subsequent chromatographic separation and deprotection of intermediates 2a-d afforded the desired target derivatives 2e-h. Product 3b was obtained by a similar acylation of 9-(3,5-O-isopropylidene-beta-D-xylofuranosyl)adenine with N-(benzyloxycarbonyl)-L-phenylalanine, followed by deblocking. The NMR spectra of 2' and 3' isomers of stereoisomers 2a and 2b are discussed. Compounds 2g and 3b are both substrates and inhibitors of Escherichia coli ribosomal peptidyltransferase, although the activity of 3b is low. Derivatives 2e,f,h do not accept AcPhe from N-AcPhe-tRNA in a peptidyltransferase-catalyzed reaction, but they inhibit the puromycin reaction in the same system. The order of inhibitory activity is 2e greater than 2f greater than 2h. The implications of these findings for the mechanism of peptidyltransferase and comparison of the latter with the action of chymotrypsin are discussed.

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