Biophysics The energy relay : A proofreading scheme based on dynamic cooperativity and lacking all characteristic symptoms of kinetic proofreading in DNA replication and protein synthesis ( ribosomes / tRNA / accuracy / mutagenesis )

Abstract

A mechanism for proofreading -biosynthetic processes requiring high accuracy is described. The previously understood "kinetic proofreading" mechanism of enhancing accuracy has distinguishing characteristics such as the nonstoichiomietric use of substrate or cosubstrate that have allowed its identification in aspects of DNA and protein synthesis. The proofreading scheme developed here, though generically related, lacks all the previous identifying features. A DNA polymerase proofreading in this manner need neither generate dNMP nor have a 3'-..5' exonuclease activity. Protein synthesis could beproofread even with stoichiometric GTP consumpton or without elongation -factor Tu-GTP. The kinetic scheme that generates this proofreading makes use of an "'energy elay" from previous substrate molecules and is a representative of a class of nonequilibrium processe's displyn dyamccoprativity. This proofreading mechanism has its own identifying characteristics, which are sufficiently subtle that they would have generally escaped notice or defied interpretation. The ability of an enzyme to discriminate between correct and incorrect substrates is based on energy differences between these substrates when bound to the enzyme in appropriate transition states. Simple Michaelis-Menten enzymes have a finite energy difference AGt between such states for similar substrates and thus must make errors at least a finite fraction fo = e of the time when presented with equal concentrations of both substrates (1, 2). Errors can represent a serious limitation on the gbiliity to process biochemical information, and cells make use of many error-reducing and errorcorrecting mechanisms to keep errors in DNA, RNA, and protein synthesis at low levels. Kinetic proofreading is the simplest general mechanism of error prevention at the molecular level (2, 3) beyond a bruteforce increase of AGt (and concomitant reduction of f0) by precise stereochemical constraints. In cases in which the enzyme does not [or even in principle cannot (1, 4)] increase the simple discrimination energy AGt, proofreading can nonetheless be used to reduce errors far below fo at the cost of an additional expenditure of energy (2, 5-7). Kinetic proofreading schemes have readily identified hallmarks (1-3). From these, it now appears that the "editing" of some prokaryotic DNA polymerases (8-11), the operation of several of the aminoacyl tRNA synthetases (12-14), and the use of elongation factor Tu-GTP in protein synthesis (15-17) and codon recognition all involve the same basic proofreading scheme and branched pathway. Yet puzzles still abound in questions of accuracy. For example, if the easiest way to gain accuracy beyondfo is by kinetic proofreading, why do few eukaryotic DNA polymerases show the hallmarks of such a process? Are the requisite activities simply lost in purification, or are they for some reason not needed? Are the replicative assemblies and the variety of proteins used in DNA or RNA polymerization or protein synthesis increasing accuracy by improving the stereochemistry, or merely by producing kinetics that allow available stereochemistry to be expressed? Or in this welter of structural complication may new phenomena be going on? Such questions form the background of the present work. The "energy relay" is a mode of enzyme operation in which the recent past can be "remembered" by an enzyme in a dissipative system. This memory can be used to obtain useful and novel results in enzyme properties. Its physical basis is the nonequilibrium populations generated in a driven kinetic system displaying multiple conformations. We will use this mechanism to produce a proofreading scheme that, though conceptually a form of kinetic proofreading, lacks all the conventional hallmarks of such proofreading. This proofreading through an energy relay and dynamic cooperativity may be taking place in polymerases, replicons, etc., where simple kinetic proofreading is believed not to occur because its identifying characteristics are absent. It may also occur in addition to simple kinetic proofreading. The hallmarks of elementary kinetic proofreading Kineticproofreading is a general means of obtaining higher accuracy from a given discrimination energy AGt between correct and incorrect substrates (1-3). The topology of the reaction pathway is different from that of a nonproofreading enzyme in that a branched reaction pathway is essential. The simple E+ S ITES I (ES)t..."E Ps I1] Michaelis-Menten pathwaty is not branched-a substrate molecule bound to the enzyme is either released as product Ps or released as S with no change in it or any other cosubstrate. Such a pathway cannot proofread (18). The elementary kinetic proofreading process demands a branched pathway, as for example (2)

Cite this paper

@inproceedings{HopfieldBiophysicsTE, title={Biophysics The energy relay : A proofreading scheme based on dynamic cooperativity and lacking all characteristic symptoms of kinetic proofreading in DNA replication and protein synthesis ( ribosomes / tRNA / accuracy / mutagenesis )}, author={John J. Hopfield} }