The transition state for formation of the peptide bond in the ribosome

Abstract

Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3′ end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, E a , is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be ≈18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2′ OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments.

The transition state for peptide bond formation reveals the ribosome as a water trap

“…The presence of this water molecule had been predicted by molecular dynamics (MD) simulations (15, 17) and was later confirmed in a highresolution crystal structure of the RAP TS analog bound to the PTC (1). None of the other proposed mechanisms (26,27) have yielded reasonable energetics in calculations (15,28).Third, there is another water molecule that is in a perfect position to stabilize any developing negative charge in the transition state on the ester carbonyl oxygen of the P-site substrate. Simulations have predicted this water molecule to be hydrogen bonded to the carbonyl oxygen (15, 17), and it was subsequently observed experimentally (1,29).…”

“…The presence of this water molecule had been predicted by molecular dynamics (MD) simulations (15, 17) and was later confirmed in a highresolution crystal structure of the RAP TS analog bound to the PTC (1). None of the other proposed mechanisms (26,27) have yielded reasonable energetics in calculations (15,28).…”

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

“…Consistently, quantum mechanical calculations, based on D50S structural data, indicated that the transition state (TS) of this reaction, namely peptide bond formation, is formed during the rotatory motion and is stabilized by hydrogen bonds with rRNA nucleotides (Gindulyte et al 2006) and is located between the A-and the P-sites at a position similar to that found experimentally in the crystal structure of a complex made of the large subunit from a ribosome from a different source, H50S, with a chemically designed TS analogue (Schmeing et al 2005a). The correlation between the rotatory motion and amino acid polymerization rationalizes the apparent contradiction associated with the location of the growing protein chain.…”

“…The structure of the large ribosomal subunit from D. radiodurans (D50S) in complexes with a substrate analogue mimicking the A-site tRNA part interacting with the large subunit, called ASM, advanced the comprehension of peptide bond formation by showing that ribosomes position their substrates in stereochemistry suitable for peptide bond formation, thus providing the machinery for peptide bond formation and tRNA translocation Agmon et al 2005). Furthermore, the ribosomal architecture, which facilitates positional catalysis of peptide bond formation, promotes substrate-mediated chemical acceleration in accord with the requirement of full-length tRNAs for rapid and smooth peptide bond formation, observed by various methods, including the use of chemical (Weinger et al 2004;Brunelle et al 2006;Weinger & Strobel 2006), mutagenesis (Sato et al 2006), computational (Sharma et al 2005;Gindulyte et al 2006;Trobro & Aqvist 2006) and kinetic procedures (Beringer et al 2005;Wohlgemuth et al 2006;Rodnina et al 2007). The current consensus view is consistent with ribosomal positional catalysis that allows for chemical catalysis by its P-site tRNA substrate.…”

“…At the A-and P-sites, the tRNA anticodon loops interact with the mRNA on the small subunit, and the acceptor stem with the aminoacylated or peptidylated 3 0 end is located on the large subunit. A-to P-site tRNA translocation comprises two highly correlated motions: sideways shift and a ribosomal navigated rotatory motion (figure 2; Agmon et al 2003Agmon et al , 2005Agmon et al , 2006Agmon et al , 2009Bashan et al 2003;Sato et al 2006;Bashan & Yonath 2008b), during which peptide bonds are being formed (Gindulyte et al 2006). This process also involves the translocation of the tRNA 3 0 end from the A-to the P-site, the detachment of the P-site tRNA from the growing polypeptide chain, the passage of the deacylated tRNA molecule to the E-site and its subsequent release.…”