Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.     

Peptide bond formation mediated by substrate mimetics. Structure-guidedoptimization of trypsin for synthesis.

Substrate mimetics are excellent tools for protease-mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino-acid-containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site-directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino-acid-containing peptides and substrate mimetics as the reactants. Finally, computer-assisted protein-ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.     

Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis.

A soluble protein EF-P (elongation factor P) from Escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70S ribosomes in vitro. Based on the partial amino acid sequence of EF-P, 18- and 24-nucleotide DNA probes were synthesized and used to screen lambda phage clones from the Kohara Gene Bank. The entire EF-P gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the E.coli genome. Two DNA fragments, 3.0 and 0.78 kilobases in length encompassing the gene, were isolated and cloned into pUC18 and pUC19. Partially purified extracts from cells transformed with these plasmids overrepresented a protein which co-migrates with EF-P upon SDS polyacrylamide gel electrophoresis, and also exhibited increased EF-P mediated peptide-bond synthetic activity. Based on DNA sequence analysis of this gene, the EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,447. The sequence and chromosomal location of EF-P establishes it as a unique gene product.     

Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix.

RNA minihelices that recreate the amino acid acceptor domain of the two-domain L-shaped tRNA molecule are substrates for specific aminoacylation by tRNA synthetases. Some lines of evidence suggest that this domain arose independently of and predated the second, anticodon-containing domain. With puromycin and a minihelix charged with alanine, we show here efficient 50S ribosome catalyzed peptide synthesis. The aminoacyl minihelix is as active as aminoacyl tRNA in the synthetic reaction. The high efficiency of the charged minihelix is due to a relatively strong interaction with the 50S particle. In contrast, an aminoacyl RNA fragment that recreates the 3'-side of the tRNA acceptor stem has a much weaker interaction with the 50S particle. These results are consistent with the minihelix domain being the major loci for tRNA interactions with the 50S ribosome. They may also have implications for the historical development of RNA-based systems of peptide synthesis.     

Peptide bond formation on the ribosome: structure and mechanism

The peptidyl transferase reaction on the ribosome is catalyzed by RNA. Pre-steady-state kinetic studies using Escherichia coli ribosomes suggest that catalysis (>10(5)-fold overall acceleration) is, to a large part, a result of substrate positioning, in agreement with crystal structures of large ribosomal subunits with bound substrate or product analogs. The rate of peptide bond formation is inhibited approximately 100-fold by protonation of a single ribosomal group with a pK(a) of 7.5, indicating general acid-base catalysis and/or a pH-dependent conformational change within the active site. According to the kinetics of mutant ribosomes, these effects may be attributed to a candidate catalytic base (A2451) suggested by the crystal structure.     

Peptide bond formation does not involve acid-base catalysis by ribosomal residues

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).     

Peptide bond formation by the industrial protease, neutrase, in organic media

Neutrase, co-deposited with sorbitol on to polyamide, synthesised several N a -protected dipeptide derivatives in acetonitrile with a buffer concentration of 4% (v/v). Reaction yields between 95-99% were obtained for all the dipeptides with the exception of Fmoc-Hyp-Leu-NH 2 (77%) and Z-Val-Leu-NH 2 (9%).     

Transacylation rates of (aminoacyl)adenosine moiety at the 3'-terminus of aminoacyl transfer ribonucleic acid

The rates of migration of the aminoacyl group (transacylation) between 2'-O-(aminoacyl)-tRNA and 3'-O-(aminoacyl)-tRNA were studied by the nuclear magnetic resonance (NMR) analyses of 3'-terminal fragment models, with regard to the significance of transacylation in the process of protein biosynthesis. 2'(3')-O-L-Alanyladenosine, -valyladenosine, -isoleucyladenosine, -phenylalanyladenosine, and -methionyladenosine, and 2'(3')-O-L-phenylalanyladenosine 5'-phosphate and methionyladenosine 5'-phosphate were chemically synthesized, and the rates of transacylation in deuterated buffer were directly measured by the NMR saturation transfer method. The dependences of transacylation rates on p2H and temperature were analyzed. The results indicate that the transacylation rates are significantly affected by the ionization states of the alpha-amino group of the amino acid moiety but not by the presence of the 5'-phosphate group of the adenylate moiety. The second-order rate constants for the base-catalyzed transacylation reactions were also determined for the ionized form (with alpha-N2H3+ group) of (aminoacyl)adenosines. The transacylation rates of (aminoacyl)adenosines in 1H2O solution at p1H 7.3 and 37 degrees C (intracellular environment) were evaluated as 3-11 s-1 for the 2' leads to 3' transacylation and 1-4 s-1 for the 3' leads to 2' transacylation, indicating that the transacylation rate of free aminoacyl-tRNA is slower than the overall rate of polypeptide chain elongation per ribosome. This suggests the presence of some enzymatic factor for enhancing the transacylation rates of aminoacyl-tRNAs in the polypeptide chain elongation process in vivo.     

Peptide bond synthesis: function of the efp gene product

The efp gene encodes a protein that is essential for the growth and for the viability of Escherichia coli cells. Interruption of this gene results in cell death due to a defect in protein synthesis. We report here that the EFP protein, encoded by the efp gene, is required for in vitro reconstitution of polypeptide synthesis in a system programmed by a native template which contains each of the purified initiation factors, IF1, IF2, IF3; the elongation factors, EFTu, EFTs and EFG, and a protein called W that is required to eject tRNAs from ribosomes. The EFP protein is required for enhancing the rate and the extent of synthesis in the presence of all of the above factors. The EFP protein stimulates synthesis of poly(Phe) programmed with poly(rU) only if N-acetyl Phe-tRNA initiates the reactions under conditions that foster the dissociation of the 70S ribosome. Study of the ability of the ribosome to synthesize a number of fMet-initiated dipeptides from CCA amino acyl acceptors suggests that EFP acts to promote synthesis with acceptors that are poor donors for the the reconstituted peptidyl transferase.     

3-Ribosyl-6-methyluracil as a phosphate acceptor in the synthesis of the internucleotide bond catalyzed by pancreatic ribonuclease.

The synthesis of uridylyl-(3'-5')-3-ribosyl-6-methyluracil (UprmU) catalyzed by pancreatic ribonuclease (EC 3.4.1.22) has been performed using uridine 2', 3'-cyclic phosphate (U greater than p) as phosphate donor and 3-ribosyl-6-methyluracil (rmU) as phosphate acceptor. The rate of synthesis of UprmU is much higher than that of uridylyl-(3'-5')-uridine (UpU) in a control experiment under the same conditions with uridine as acceptor. The yields of UpU and UprmU were 20 and 17% respectively. The competitive hydrolysis of the initial U greater than p also proceeds faster when rmU is used as the acceptor. The relationship between the conformation of this nucleoside and its acceptor activity in the enzymatic synthesis of the internucleotide bond is discussed.     

Modulation of protein synthesis by nerve growth factor.

The effects of nerve growth factor (NGF) and two analogs of cAMP have been studied in the PC12 line of rat pheochromocytoma cells. We have used two-dimensional gel electrophoresis to detect more than 800 proteins from control and NGF-treated cells. Of these proteins, none were qualitatively repressed in response to NGF, and no new proteins appeared after NGF treatment. Visual inspection of the gels showed that approximately 5% of the proteins were detectably increased or decreased in rate of synthesis by NGF, and each of these changes was mimicked by both cAMP analogs. The two-dimensional gel data were further analyzed by a computerized scanning system. This analysis has revealed many significant changes that are smaller than those detected by eye. Approximately 25 to 30% of the proteins analyzed were found to be altered in rate of synthesis by 30% or more. Statistical analysis has shown that the response to NGF and the response to dibutyryl cAMP are highly correlated, even down to changes as small as 30%. No proteins were found to be significantly altered by both dibutyryl cAMP and 8-bromo cAMP, but not by NGF. These results show that NGF causes only quantitative modulations of protein synthesis in PC12 cells, and these data strongly suggest that the response of PC12 cells to NGF is mediated by cAMP.