Kinetic proofreading (or kinetic amplification) is a mechanism for error correction in biochemical reactions, proposed independently by John Hopfield (1974) and Jacques Ninio (1975). Kinetic proofreading allows enzymes to discriminate between two possible reaction pathways leading to correct or incorrect products with an accuracy higher than what one would predict based on the difference in the activation energy between these two pathways. [1] [2]
Increased specificity is obtained by introducing an irreversible step exiting the pathway, with reaction intermediates leading to incorrect products more likely to prematurely exit the pathway than reaction intermediates leading to the correct product. If the exit step is fast relative to the next step in the pathway, the specificity can be increased by a factor of up to the ratio between the two exit rate constants. (If the next step is fast relative to the exit step, specificity will not be increased because there will not be enough time for exit to occur.) This can be repeated more than once to increase specificity further.
As an analogy, if we have a medicine assembly line sometimes produces empty boxes, and we are unable to upgrade the assembly line, then we can increase the ratio of full boxes over empty boxes (specificity) by placing a giant fan at the end. Empty boxes are more likely to be blown off the line (a higher exit rate) than full boxes, even though both kinds' production rates are lowered. By lengthening the final section and adding more giant fans (multistep proofreading), the specificity can be increased arbitrarily, at the cost of decreasing production rate.
In protein synthesis, the error rate is on the order of . This means that when a ribosome is matching anticodons of tRNA to the codons of mRNA, it matches complementary sequences correctly nearly all the time. Hopfield noted that because of how similar the substrates are (the difference between a wrong codon and a right codon can be as small as a difference in a single base), an error rate that small is unachievable with a one-step mechanism. Both wrong and right tRNA can bind to the ribosome, and if the ribosome can only discriminate between them by complementary matching of the anticodon, it must rely on the small free energy difference between binding three matched complementary bases or only two.
A one-shot machine which tests whether the codons match or not by examining whether the codon and anticodon are bound will not be able to tell the difference between wrong and right codon with an error rate less than unless the free energy difference is at least 9.2kT, which is much larger than the free energy difference for single codon binding. This is a thermodynamic bound, so it cannot be evaded by building a different machine. However, this can be overcome by kinetic proofreading, which introduces an irreversible step through the input of energy. [3]
Another molecular recognition mechanism, which does not require expenditure of free energy is that of conformational proofreading. The incorrect product may also be formed but hydrolyzed at a greater rate than the correct product, giving the possibility of theoretically infinite specificity the longer you let this reaction run, but at the cost of large amounts of the correct product as well. (Thus there is a tradeoff between product production and its efficiency.) The hydrolytic activity may be on the same enzyme, as in DNA polymerases with editing functions, or on different enzymes.
Hopfield suggested a simple way to achieve smaller error rates using a molecular ratchet which takes many irreversible steps, each testing to see if the sequences match. At each step, energy is expended and specificity (the ratio of correct substrate to incorrect substrate at that point in the pathway) increases.
The requirement for energy in each step of the ratchet is due to the need for the steps to be irreversible; for specificity to increase, entry of substrate and analogue must occur largely through the entry pathway, and exit largely through the exit pathway. If entry were an equilibrium, the earlier steps would form a pre-equilibrium and the specificity benefits of entry into the pathway (less likely for the substrate analogue) would be lost; if the exit step were an equilibrium, then the substrate analogue would be able to re-enter the pathway through the exit step, bypassing the specificity of earlier steps altogether.
Although one test will fail to discriminate between mismatched and matched sequences a fraction of the time, two tests will both fail only of the time, and N tests will fail of the time. In terms of free energy, the discrimination power of N successive tests for two states with a free energy is the same as one test between two states with a free energy .
To achieve an error rate of requires several comparison steps. Hopfield predicted on the basis of this theory that there is a multistage ratchet in the ribosome which tests the match several times before incorporating the next amino acid into the protein.
Biochemical processes that use kinetic proofreading to improve specificity implement the delay-inducing multistep ratchet by a variety of distinct biochemical networks. Nonetheless, many such networks result in the times to completion of the molecular assembly and the proofreading steps (also known as the first passage time) that approach a near-universal, exponential shape for high proofreading rates and large network sizes. [10] Since exponential completion times are characteristic of a two-state Markov process, this observation makes kinetic proofreading one of only a few examples of biochemical processes where structural complexity results in a much simpler large-scale, phenomenological dynamics.
The increase in specificity, or the overall amplification factor of a kinetic proofreading network that may include multiple pathways and especially loops is intimately related to the topology of the network: the specificity grows exponentially with the number of loops in the network. [11] [12] An example is homologous recombination in which the number of loops scales like the square of DNA length. [5] [6] The universal completion time emerges precisely in this regime of large number of loops and high amplification. [11]
Enzymes are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and the field of pseudoenzyme analysis recognizes that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties.
The genetic code is the set of rules used by living cells to translate information encoded within genetic material into proteins. Translation is accomplished by the ribosome, which links proteinogenic amino acids in an order specified by messenger RNA (mRNA), using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time. The genetic code is highly similar among all organisms and can be expressed in a simple table with 64 entries.
Protein biosynthesis is a core biological process, occurring inside cells, balancing the loss of cellular proteins through the production of new proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences.
Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.
In biochemistry, Michaelis–Menten kinetics, named after Leonor Michaelis and Maud Menten, is the simplest case of enzyme kinetics, applied to enzyme-catalysed reactions of one substrate and one product. It takes the form of a differential equation describing the reaction rate to , the concentration of the substrate A. Its formula is given by the Michaelis–Menten equation:
In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.
Helicases are a class of enzymes thought to be vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two hybridized nucleic acid strands, using energy from ATP hydrolysis. There are many helicases, representing the great variety of processes in which strand separation must be catalyzed. Approximately 1% of eukaryotic genes code for helicases.
Pyruvate kinase is the enzyme involved in the last step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP. Pyruvate kinase was inappropriately named before it was recognized that it did not directly catalyze phosphorylation of pyruvate, which does not occur under physiological conditions. Pyruvate kinase is present in four distinct, tissue-specific isozymes in animals, each consisting of particular kinetic properties necessary to accommodate the variations in metabolic requirements of diverse tissues.
Biosynthesis, i.e., chemical synthesis occurring in biological contexts, is a term most often referring to multi-step, enzyme-catalyzed processes where chemical substances absorbed as nutrients serve as enzyme substrates, with conversion by the living organism either into simpler or more complex products. Examples of biosynthetic pathways include those for the production of amino acids, lipid membrane components, and nucleotides, but also for the production of all classes of biological macromolecules, and of acetyl-coenzyme A, adenosine triphosphate, nicotinamide adenine dinucleotide and other key intermediate and transactional molecules needed for metabolism. Thus, in biosynthesis, any of an array of compounds, from simple to complex, are converted into other compounds, and so it includes both the catabolism and anabolism of complex molecules. Biosynthetic processes are often represented via charts of metabolic pathways. A particular biosynthetic pathway may be located within a single cellular organelle, while others involve enzymes that are located across an array of cellular organelles and structures.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
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Flap endonuclease 1 is an enzyme that in humans is encoded by the FEN1 gene.
The term proofreading is used in genetics to refer to the error-correcting processes, first proposed by John Hopfield and Jacques Ninio, involved in DNA replication, immune system specificity, and enzyme-substrate recognition among many other processes that require enhanced specificity. The proofreading mechanisms of Hopfield and Ninio are non-equilibrium active processes that consume ATP to enhance specificity of various biochemical reactions.
Conformational proofreading or conformational selection is a general mechanism of molecular recognition systems, suggested by Yonatan Savir and Tsvi Tlusty, in which introducing an energetic barrier - such as a structural mismatch between a molecular recognizer and its target - enhances the recognition specificity and quality. Conformational proofreading does not require the consumption of energy and may therefore be used in any molecular recognition system. Conformational proofreading is especially useful in scenarios where the recognizer has to select the appropriate target among many similar competitors. Proteins evolve the capacity for conformational proofreading through fine-tuning their geometry, flexibility and chemical interactions with the target.
This glossary of cellular and molecular biology is a list of definitions of terms and concepts commonly used in the study of cell biology, molecular biology, and related disciplines, including genetics, biochemistry, and microbiology. It is split across two articles:
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