An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA

Parallel to the amide bond formation, the hydrolysis of the active esters of α/β-amino acids, as an unwanted side reaction limiting coupling efficacy, is studied.

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Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation

Biochemical Journal ◽

10.1042/bj1560015 ◽

1976 ◽

Vol 156 (1) ◽

pp. 15-23 ◽

Cited By ~ 10

Author(s):

L Montanaro ◽

S Sperti ◽

G Testoni ◽

A Mattioli

Keyword(s):

Rat Liver ◽

Bond Formation ◽

Elongation Factor ◽

Adenosine Diphosphate ◽

Peptide Bond ◽

Native Enzyme ◽

Peptide Bond Formation ◽

Elongation Factor 2 ◽

Hydrolysis Of ◽

Liver Ribosomes

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.

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N-Methylamino Acids in Peptide Synthesis. IV. Racemization and Yields in Peptide-bond Formation

Canadian Journal of Chemistry ◽

10.1139/v73-386 ◽

1973 ◽

Vol 51 (15) ◽

pp. 2562-2570 ◽

Cited By ~ 61

Author(s):

John R. McDermott ◽

N. Leo Benoiton

Keyword(s):

Bond Formation ◽

Addition Product ◽

Peptide Bond ◽

Triethylamine Hydrochloride ◽

Peptide Bond Formation ◽

Ester Method ◽

High Yields ◽

Excess Base ◽

Hydrolysis Of ◽

Model Peptides

The racemization of an N-methylamino-acid residue during peptide-bond formation and mixed-anhydride activation has been investigated using Ala-MeLeu-Gly and Ala-MeLeu as model peptides. The results were compared with those for Ala-Leu-Gly, Ala-Leu, and Ala-Pro. The extents of racemization were determined by analysis of the diastereomeric products of the reactions after deprotection, using an amino-acid analyzer. Extensive racemization was detected after the hydrolysis of the mixed anhydrides of Bz-MeLeu, Z-Ala-MeLeu, and Z-Ala-Leu, but not of Boc-Ala-Pro and Z-MeIle. Significant racemization (2.8–39%) was observed when Z-Ala-MeLeu was coupled with Gly-OBzl by various methods in the presence of salts such as triethylamine hydrochloride or p-toluenesulfonate. Only coupling through the N-hydroxysuccinimide (HONSu) ester gave stereochemically pure product. In the absence of salt, less racemization was observed, but only couplings using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and N,N′-dicyclohexylcarbodiimide–HONSu gave essentially pure products. Polar solvents promoted racemization, but excess base did not. Chemical evidence that the racemization intermediate is an oxazolium-5-oxide has been obtained by trapping the intermediate as an addition product (a pyrrole) in 85% yield.The yields obtained by various coupling methods have been determined for several model peptides. Couplings at the carboxyl group of an N-methylamino acid gave high yields only in the absence of salt, except for coupling by the HONSu ester method. Couplings to an N-methylamino group gave high yields, except for coupling by the p-nitrophenyl ester method. Couplings to Ala-MeLeu-OBu′ gave higher yields than couplings to Ala-MeLeu-OBzl, presumably due to piperazine-dione formation by the latter.

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