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ScienceWeek
CHEMISTRY: CHEMICAL SPACE AND BIOLOGY
The following points are made by Christopher M. Dobson (Nature 2004 432:824):
1) Living systems have evolved over several billion years to carry out carefully controlled chemistry in an aqueous environment at temperatures almost exclusively between zero and 100 °C. Under these conditions and unaided, many of the chemical reactions that are essential to life would not occur at perceptible rates, and most would not result in specific and reproducible products. Enzymes, along with other proteins and some nucleic acids, are used by natural biological systems to achieve this control; these macromolecules are responsible for the synthesis, transport and degradation of virtually every chemical compound in the biological environment[1].
2) However, the chemical compounds used by biological systems represent a staggeringly small fraction of the total possible number of small carbon-based compounds with molecular masses in the same range as those of living systems (that is, less than about 500 daltons). Some estimates of this number are in excess of 10^(60) [2]. The simplest living organisms can function with just a few hundred different types of such molecules, and fewer than 100 account for nearly the entire molecular pool[3,4].
3) Moreover, it seems that the total number of different small molecules within our own bodies could be just a few thousand[4]. So it is clear that at least in terms of numbers of compounds, "biologically relevant chemical space" is only a minute fraction of complete "chemical space". It is remarkable that so many complex processes can be carried out with such a limited number of molecules, and that biological chemistry can be so rich and diverse despite the relatively limited range of reactions that seem to have been exploited during the evolution of living systems.
4) Similarly, as revealed by the recent triumphs of a variety of international sequencing projects, the genomes of the simplest living systems encode the sequences of less than 1000 different proteins and the human genome about 100 times more[5] -- numbers that are minute when compared with the total number of proteins that could theoretically exist. As there are 20 different types of amino acid and the average size of a natural protein is about 300 residues, this number is a staggering 20^(300) or more than 10^(390), and if only a single molecule of each of these polypeptides were to be produced, their combined mass would vastly exceed that of the known universe. Natural proteins are therefore also a very select group of molecules.
5) The characteristics of this select group of natural proteins are linked to those of the small molecules that are used in living systems, and to those of the relatively small number of synthetic small molecules that we have developed into drugs. Understanding this link will help us answer the question of how we can best use the powerful new methods that are emerging to probe biological systems, both to understand the fundamental processes of life and to develop new strategies to treat disease.
References (abridged):
1. Fersht, A. R. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding (W. H. Freeman, New York, 1999)
2. Bohacek, R. S., McMartin, C. & Guida, W. C. The art and practice of structure-based drug design: a molecular modelling perspective. Med. Res. Rev. 16, 3-50 (1996)
3. Luria, S. E., Gould, S. J. & Singer, S. A View of Life (Benjamin/Cummings, Menlo Park, California, 1981)
4. Goto, S., Okuno, Y., Hattori, M., Nishioka, T. & Kanehisa, M. LIGAND: database of chemical compounds and reactions in biological pathways. Nucleic Acids Res. 30, 402-404 (2002)
5. Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature 409, 806-921 (2001)
Nature http://www.nature.com/nature
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PROTEIN CHEMISTRY: COMPUTATIONAL DESIGN OF AN ACTIVE ENZYME
The following points are made by M.A. Dwyer et al (Science 2004 304:1967):
1) Enzymes are among the most proficient catalysts known (1), and they catalyze a wide variety of reactions in aqueous solutions under ambient conditions with exquisite selectivity and stereospecificity (2,3). The rational design of enzymes has tremendous practical potential for developing novel synthetic biochemical pathways (4,5), but presents a formidable challenge and is one of the most stringent tests for understanding protein chemistry.
2) The authors present structure-based computational design techniques that predict mutations for the construction of catalytically active sites in proteins of known structure. Using these methods, the authors converted ribose-binding protein into analogs (NovoTims) of the glycolytic enzyme triose phosphate isomerase. Several NovoTims exhibit rate enhancements of about 10^(5) to 10^(6) and are biologically active, as seen in their support of the growth of Escherichia coli under gluconeogenic conditions.
3) Triose phosphate isomerase (TIM) is an essential component of the Embden-Meyerhof pathway, interconverting dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). In glycolysis, TIM channels these two triose phosphate products of aldolase into pyruvate; in gluconeogenesis, TIM ensures that both substrates are supplied to aldolase. The isomerization reaction involves two successive proton exchanges and is considered an archetype for proton transfer chemistry, which is central to many enzyme mechanisms. Extensive studies support a mechanism whereby a carboxylate abstracts the DHAP pro-R proton at C1 to form a cis-enediol(ate) intermediate, followed by imidazole-mediated proton transfer between the C1 and C2 oxygens, yielding GAP. The C1 proton equilibrium constant (pKa) of ~18 imposes a large barrier to proton abstraction, which is overcome by a low-barrier hydrogen bond that requires precise functional group alignment.
4) In summary: Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. The authors present and experimentally validate the computational design of enzyme activity in proteins of known structure. The authors have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10^(5)- to 10^(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The authors suggest that the inherent generality of the design method indicates that many enzymes can be designed by this approach.
References (abridged):
1. R. Wolfenden, M. J. Snider, Acc. Chem. Res. 34, 938 (2001)
2. S. J. Benkovic, S. Hammes-Schiffer, Science 301, 1196 (2003)
3. M. Garcia-Viloca, J. Gao, M. Karplus, D. G. Truhlar, Science 303, 186 (2004)
4. D. N. Bolon, C. A. Voigt, S. L. Mayo, Curr. Opin. Chem. Biol. 6, 125 (2002)
5. D. Hilvert, Annu. Rev. Biochem. 69, 751 (2000)
Science http://www.sciencemag.org
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BIOCHEMISTRY: ON ENZYME CATALYSIS
The following points are made by M. Garcia-Viloca et al (Science 2004 303:186):
1) Enzyme catalysis, which can produce rate accelerations as large as a factor of 10^(19) (1), involves molecular recognition at the highest level of development. The catalysis of many proton-transfer reactions, for example, requires the recognition of a change in a CH bond length of about 0.5 in going from the reactant to the transition state. In 1946, before structural information was available, Linus Pauling (1901-1994) proposed (2) that enzymes can accelerate rates because they bind the transition state better than the substrate and thereby lower the activation energy.
2) This key concept in enzyme catalysis can now be augmented by a detailed description of the sources of enzymatic rate enhancements based on developments in transition state theory, the availability of structural, kinetic, and thermodynamic data, and the insights provided by computer simulations. An increasing number of articles propose a variety of origins for enzyme catalysis described by terms such as correlated conformational fluctuations, dynamical and nonequilibrium effects, electrostatic pre-organization, entropic guidance, fluctuating barrier height, near-attack configurations, reactant destabilization, and tunneling.
3) In an insightful paper published in 1978 (3), which is as valid today as it was then, Schowen wrote, in accord with Pauling's original insight, "...the entire and sole source of catalytic power [of enzymes] is the stabilization of the transition state..." The authors introduce modern concepts of transition state theory that led to a somewhat modified and more detailed description of the role of the transition state and use it as the framework for describing our present understanding of how enzymes "work". The authors' restatement of the key premise is that "the entire and sole source of the catalytic power of enzymes is due to the lowering of the free energy of activation and any increase in the generalized transmission coefficient, as compared to that of the uncatalyzed reaction."
4) In summary: Advances in transition state theory and computer simulations are providing new insights into the sources of enzyme catalysis. Both lowering of the activation free energy and changes in the generalized transmission coefficient (recrossing of the transition state, tunneling, and nonequilibrium contributions) can play a role. A framework for understanding these effects is presented by the authors, and the contributions of the different factors, as illustrated by specific enzymes, are identified and quantified by computer simulations.(4,5)
References (abridged):
1. R. Wolfenden, M. J. Snyder, Acc. Chem. Res. 34, 938 (2001)
2. L. Pauling, Chem. Eng. News 24, 1375 (1946)
3. R. L. Schowen, in Transition States of Biochemical Processes, R. D. Gandour, R. L. Schowen, Eds. (Plenum, New York, 1978), p77.
4. An historical overview of mechanistic enzymology, which serves as an excellent complement to the present analysis, was published recently by Bugg (2001). He points out that many enzymologists continue to search for the "elusive additional mechanisms by which enzymes may achieve high rates of catalysis". The thesis of the present article is that modern simulations of transition states are a powerful tool for discovering these mechanisms and that all such mechanisms can be understood in terms of various contributions to a specific equation from transition state theory relating the rate constant for a reaction as a function of the temperature.
5. H. Eyring, A. E. Stern, Chem. Rev. 24, 2 (1939)
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