Issue Date: November 29, 2004
RIBOSOME MECHANISM IS A PUZZLER
The underlying mechanism of the ribosome, the RNA-protein complex that catalyzes peptide bond formation leading to protein synthesis, is becoming clearer, thanks to recent efforts by several groups.
"The ribosome community is today, for the first time in its history, having to seriously confront issues that have been on the front burner for enzymologists ever since the structure of lysozyme was solved in the mid-1960s--making the connection between the structure of an enzyme and the mechanism by which it catalyzes the reaction that occurs in its active site," says Yale University professor of chemistry and of molecular biophysics and biochemistry Peter B. Moore.
The ribosome is a giant, complicated molecular machine. Both experimental and theoretical studies of it are fraught with potential pitfalls, uncertainties, and difficulties. A consensus on its mechanism has not yet been reached, and some studies have come to seemingly contradictory conclusions. However, scientists are getting closer to fathoming its mechanistic secrets.
Three basic mechanisms are possible: First, the ribosome may have a primarily passive, entropic mechanism, in which it lowers the activation energy (raises the ground state) of protein synthesis by bringing substrates together in an appropriate orientation so that they can react more easily. Second, the ribosome may achieve most of its accelerating power by an active, enthalpic mechanism, in which it lowers the activation energy of the reaction by interacting chemically with substrates or with the transition state (the high-energy complex in which a reaction becomes committed to creating its product). And third, the mechanism may include a substantial degree of both entropic and enthalpic contributions.
In 1980, professor of molecular genetics Knud H. Nierhaus of the Max Planck Institute of Molecular Genetics, Berlin; chemistry professor Barry S. Cooperman of the University of Pennsylvania; and a coworker proposed that the ribosome's ability to properly orient two aminoacylated transfer RNA substrates in its peptidyl transferase center (active site) was sufficient to account for all of its catalytic power. This suggested a primarily entropic mechanism.
The proposal received further support in 2000 and 2001, after atomic-level X-ray crystallographic structures of the ribosome were reported by three groups--a team led by professor of molecular biophysics and chemistry and Howard Hughes Medical Institute investigator Thomas A. Steitz and his colleague Moore at Yale; a group led by Venki Ramakrishnan of the Medical Research Council Laboratory of Molecular Biology, Cambridge, England; and a group including structural biology professor Ada E. Yonath at Weizmann Institute of Science, Rehovot, Israel, and the Max Planck Research Unit for Structural & Molecular Biology, Hamburg, Germany, and François J. Franceschi of the Max Planck Institute for Molecular Genetics, Berlin.
STRUCTURAL ANALYSES by the Steitz-Moore and Yonath-Franceschi groups indicated that the ribosome's main catalytic contribution was to properly position two substrate tRNAs for the peptide-bond-forming reaction and to ease a rotatory transition that occurs in the course of the reaction. In the reaction, an aminoacyl (amino acid-carrying) tRNA in the peptidyl transferase center's aminoacyl (A) site reacts with a peptidyl tRNA in the ribosome's peptidyl (P) site and makes a concerted rotatory motion to form a new peptidyl tRNA one amino acid longer than the previous one. The structural analyses suggested that the ribosome's role in this process was primarily entropic.
This May, a similar conclusion was reached in a study by professor of chemistry, biochemistry, and biophysics Richard V. Wolfenden of the University of North Carolina, Chapel Hill; professor of physical biochemistry Marina V. Rodnina of the University of Witten/Herdecke, Germany; and coworkers. A hallmark of an enzyme's direct chemical involvement in a reaction (and thus the enthalpic character of its mechanism) is strong temperature dependence of its catalytic activity. However, a kinetic study carried out by the Wolfenden-Rodnina group demonstrated no such temperature dependence of the ribosome's activity [Proc. Natl. Acad. Sci. USA, 101, 7897 (2004)]. "That means the ribosome's effect is purely entropic," Wolfenden says. "It suggests that the ribosome acts as a mechanical matchmaker or readout device, rather than participating chemically to speed up the reaction."
Yonath says she was excited that the Wolfenden-Rodnina kinetic measurements "supported our structural results." And Steitz says that the study's findings "certainly strongly enhanced the evidence" for the then widely held belief that the majority of the rate enhancement was provided by substrate orientation. Moore is more cautious about the study's conclusions because he questions some of its methodology.
Meanwhile, calculations by chemistry professor Arieh Warshel of the University of Southern California indicate that the entropic effect found by the Wolfenden-Rodnina team is primarily due to changes in solvent entropy and not to preorganization of reactants in the ribosome's active site. Wolfenden, Rodnina, and coworkers had considered this alternative possibility and mentioned it in their paper.
Also in May, associate professor of molecular biology and genetics Rachel Green of Johns Hopkins University School of Medicine and coworkers investigated an enthalpy-based hypothesis that an active-site nucleotide in the ribosome's RNA portion might be acting as a base in the enzyme's catalytic mechanism. They found that mutating any of four key nucleotide bases close to the site of peptide bond formation has no effect on catalysis [Cell, 117, 589 (2004)].
These findings seem to rule out an enthalpic role by the active-site nucleotides. But Green notes that the results are not inconsistent with the idea that one of the ribosome's tRNA substrates could be making a significant enthalpic contribution to catalysis.
THIS MONTH, just such a substrate enthalpic effect was reported by professor of molecular biophysics and biochemistry Scott A. Strobel of Yale University and coworkers, including Green [Nat. Struct. Mol. Biol., 11, 1101 (2004)]. The Strobel-Green study showed that a 29-hydroxyl group on the ribosome's P-site tRNA substrate makes a significant chemical contribution to the catalytic reaction. When the researchers replaced that hydroxyl group with hydrogen or fluorine, the rate of ribosome-catalyzed peptide bond formation was reduced by more than four orders of magnitude, after correction for experimental effects caused by the substitutions.
Given that the ribosome normally accelerates the reaction about 107-fold, the enthalpic influence of the hydroxyl group accounts for a substantial portion of the ribosome's catalytic effect. Entropic positioning might account for the rest. Similar findings about the 29-hydroxyl group had been reported earlier by professor of biochemistry Andrea Barta of the Medical University of Vienna, Austria, and coworkers, but the Strobel-Green study is more quantitative.
"These results indicate that the ribosome utilizes substrate-assisted catalysis" and suggest "that chemical catalysis [enthalpy] and substrate alignment [entropy] both play a critical role in ribosomal protein synthesis," Strobel says.
The findings "change the mechanistic story of the ribosome yet again," he adds. "What we are uncertain about is how this 29-hydroxyl contributes to catalysis, though there are experiments under way to address this question. I think the story remains to be fully told."
"We are all struggling to understand a growing body of data that does not yet seem to make sense," Moore says. "As usual in such situations, different players have different takes on what is going on. The difficulties are the same as those that mechanistic enzymologists have confronted for decades."
Such problems are magnified and exacerbated by the ribosome's tremendous size and complexity. One thing is certain: We have not heard the last word on the mechanism of the ribosome.
- Chemical & Engineering News
- ISSN 0009-2347
- Copyright © American Chemical Society