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ScienceWeek
BIOCHEMISTRY: ON ALLOSTERIC MECHANISMS OF SIGNAL TRANSDUCTION
The following points are made by J-P. Changeux and S.J. Edelstein (Science 2005 308:1424):
1) Forty years ago, a simple model of allosteric mechanisms (indirect interactions between distinct sites), used initially to explain feedback-inhibited enzymes, was presented by Monod, Wyman, and Changeux. The elaboration of the allosteric theory spanned the years 1961 to 1967 and developed in two principal steps. The first issue involved the mechanisms by which a regulatory ligand (such as an enzyme feedback inhibitor) controls the state of activity of a biologically active site, such as an enzyme catalytic site, despite being structurally different from the active-site substrate.
2) Regulatory effectors and substrates were proposed to behave as two distinct categories of ligands, which bind to their target protein at topographically "distinct sites" [1] that mutually influence each other through a reversible conformational change. The proposal relied on the induced-fit theory of Koshland [2], which initially was developed not to explain the regulation of enzyme activity by a metabolic signal but to account for the specificity of enzyme action. This concept of indirect or "allosteric" interactions between stereospecifically distinct sites [3] differed from the classical explanations of enzyme inhibition through steric hindrance at a common binding site.
3) The second issue was raised by the analysis of the complex patterns of kinetics encountered with bacterial regulatory enzymes, particularly L-threonine deaminase and aspartate transcarbamylase. Both of these enzymes showed intertwined cooperative (homotropic) interactions between identical ligands (i.e., oxygen and hemoglobin), as well as signaling (heterotropic) interactions between different ligands (i.e., between a regulatory molecule and a substrate).
4) To deal with these issues, Monod, Wyman, and Changeux [4] proposed two unifying concepts in their 1965 "MWC" model. The first proposes that regulatory proteins have a quaternary structure (the spatial arrangements and interactions of individual polypeptide chains that together make a complex protein) with identical subunits regularly organized into finite assemblies, or "oligomers," with symmetry properties (defined in terms of axes about which one polypeptide chain with its precise three-dimensional structure can be rotated to superimpose on another chain within the same quaternary structure). The second concept postulates that to account for the observed linkage between homotropic and heterotropic interactions, the signaling oligomers undergo reversible transitions between discrete conformations, which primarily affect the quaternary organization, preserve its symmetry, and are accessible in the absence of ligand. In other words, the cooperative structural changes intrinsic to the protein molecule determine the observed cooperative binding properties.
5) Such spontaneous "conformational switches," whose states are selectively stabilized by the ligands to which they preferentially bind, contrast with the sequential, induced-fit mechanism [5] initially suggested for hemoglobin. In an induced-fit mechanism, the very binding of the ligand to its site causes a subsequent change of conformation that would be "adapted" to the particular structure of the ligand. The initial versions of the MWC theory [3,4] -- which relied on the then available structural data of Perutz for hemoglobin -- dealt with regulatory enzymes, but a plausible application to hormone nuclear receptors and gene repressors was suggested. An extension of the theory to membrane receptors, in particular to neurotransmitters, was later proposed.
References (abridged):
1. J.-P. Changeux, Cold Spring Harbor Symp. Quant. Biol. 26, 313 (1961)
2. D. E. Koshland Jr., J. Cell. Comp. Physiol. 54, 245 (1959)
3. J. Monod, J.-P. Changeux, F. Jacob, J. Mol. Biol. 6, 306 (1963)
4. J. Monod, J. Wyman, J.-P. Changeux, J. Mol. Biol. 12, 88 (1965)
5. D. E. Koshland, G. Nemethy, D. Filmer, Biochemistry 5, 365 (1966)
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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)
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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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