The articles of the conference proceedings are published in Perspectives in Science and freely accessible.
Proceedings of the
6th Beilstein ESCEC Symposium
... Celebrating the 100th Anniversary of Michaelis-Menten Kinetics
16 – 20 September 2013, Rüdesheim, Germany
Celebrating the 100th Anniversary of Michaelis-Menten Kinetics
Carsten Kettner and Martin G. Hicks
Beilstein-Institut, Frankfurt am Main, Germany
In a small research laboratory at one of Berlin’s municipal hospitals (“Am Urban”) two researchers’ very careful work laid the foundation for enzyme kinetics as a systematic field which formed the basis of modern enzymology: In 1913, Leonor Michaelis and his coworker Maud L. Menten published a paper on the concept of an affinity constant, by studying the relationship between the rate of formation of products in dependence of the concentrations of an enzyme (invertase) and its substrate at constant and controlled pH (Michaelis and Menten, 1913). The best-known outcome of this work was the Michaelis–Menten equation, together with the Michaelis constant. This term was coined by Briggs and Haldane (1925), whose work on enzyme kinetics led to the steady-state approximation assuming a negligible rate of the change of the enzyme–substrate complex compared to the rates of changes in the concentrations of both the substrate and the product. Since then enzymes have been routinely characterized by applying Michaelis and Menten’s approach and evaluation of enzymatic activities. Over the last hundred years this has allowed mechanistic models to be developed and has led to the discovery of a tremendous number of new metabolic pathways in cells and tissues.
One Hundred Years of Michaelis-Menten Kinetics
Unité de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Aix-Marseille Université, France
The year 2013 marked the centenary of the paper of Leonor Michaelis and Maud Menten (Michaelis and Menten, 1913), and the 110th anniversary of the doctoral thesis of Victor Henri (Henri, 1903). These publications have had an enormous influence on the progress of biochemistry, and are more often cited in the 21st century than they were in the 20th. Henri laid the groundwork for the understanding of enzyme mechanisms, but his experimental design was open to criticism. He reached essentially correct conclusions about the action of invertase, but he took no steps to control the hydrogen-ion concentration, and he took no account of the spontaneous mutarotation of the glucose produced in the reaction. Michaelis and Menten corrected these shortcomings, and in addition they introduced the initial-rate method of analysis, which has proved much simpler to apply than the methods based on time courses that it replaced. In this way they defined the methodology for steady-state experiments that has remained standard for 100 years.
Understanding Mechanisms of Enzyme Co-operativity:
The Importance of not being at Equilibrium
María Luz Cárdenas
Unité de Bioénergétique et Ingénierie de Protéines, CNRS-Aix Marseille Université, France
The discovery at the end of the 1950s and the beginning of the 1960s that there were enzymes like threonine deaminase and aspartate transcarbamoylase that failed to follow the expected hyperbolic behaviour predicted by the Michaelis–Menten equation, raised several questions and induced the development of mechanisms to explain this peculiar behaviour. At that time it was already known that the binding of oxygen to haemoglobin did not follow a hyperbolic curve, but a sigmoidal one, and it was thought that a similar situation probably existed for enzymes with sigmoidal kinetics. In other words, the observed kinetic behaviour was a consequence of co-operativity in the substrate binding. Two main models were postulated: those of Monod, Wyman and Changeux in 1965 and of Koshland, Némethy and Filmer in 1966. Both consider that the different conformations are in equilibrium and that there is a rapid equilibrium in the binding, which implies that co-operativity could only exist if there is more than one substrate binding site per enzyme molecule, that is, if the enzyme is an oligomer. What about monomeric enzymes, could they show kinetic co-operativity? Yes, but only through mechanisms that imply the existence of enzyme conformations that are not in equilibrium, and have different kinetic parameters. There are, in fact, very few examples of monomeric enzymes showing kinetic co-operativity with a natural substrate. The case of “glucokinase” (hexokinase D or hexokinase IV), a monomeric enzyme with co-operativity with respect to glucose, will be discussed.
Diversity in Fosfomycin Resistance Proteins
Matthew K. Thompson1, Mary E. Keithly2, Gary A. Sulikowski1,2 and Richard N. Armstrong1,2,3
1Department of Biochemistry, Vanderbilt University, Nashville, USA
2Department of Chemistry, Vanderbilt University, Nashville, USA
3Center for Structural Biology, Vanderbilt University, Nashville, USA
Certain strains of the soil microorganism Streptomyces produce an antibiotic, fosfomycin [(1 R,2 S)-epoxypropylphosphonic acid], which is effective against both Gram-positive and Gram-negative pathogens by inhibiting the first committed step in cell-wall biosynthesis. Fosfomycin resistance proteins are metallo-enzymes that are known to inactivate the antibiotic by the addition of nucleophiles such as water, glutathione (GSH), l-cysteine and bacillithiol (BSH) to the oxirane ring of the molecule. Progress in the characterisation of FosB-type fosfomycin resistance proteins found in many Gram-positive organisms has been slow. This paper provides a brief description of the diversity of fosfomycin resistance proteins in general and, more specifically, new data characterising the substrate selectivity, structure, mechanism and metal-ion dependence of FosB enzymes from pathogenic strains of Staphylococcus and Bacillus. These new findings include the high-resolution X-ray diffraction structures of FosB enzymes from Staphylococcus aureus and Bacillus cereus in various liganded states and kinetic data that suggest that Mn(II) and BSH are the preferred divalent cation and thiol substrate for the reaction, respectively. The discovery of the inhibition of the enzyme by Zn(II) led to the determination of a ternary structure of the FosB·Zn(II)·fosfomycin·l-Cys complex which reveals both substrates present in a pose prior to reaction.
Structure and Function of the Translesion DNA Polymerases and Interactions with Damaged DNA
F. Peter Guengerich, Linlin Zhao, Matthew G. Pence and Martin Egli
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, USA
Modification of DNA is a common event, due to reaction with both exogenous and endogenous factors. The resulting DNA adducts cause blockage of replicative DNA polymerases and also replication errors in cases in which the adducts can be bypassed. Translesion DNA polymerases exist in all forms of life and can replicate past bulky lesions, although with low fidelity. Our research has focused on the interactions of these polymerases with damaged DNA.
Pre-steady-state kinetic analysis has been used to develop minimum kinetic models with rate constants of (the eight) individual reaction steps in the catalytic cycle. The use of single-tryptophan mutants of Sulfolobus solfataricus Dpo4 and human (h) pol κ has led to discernment of the steps for the conformation change (associated with dNTP binding and relocation) and nucleotidyl transfer. X-ray crystal structures have been obtained for a number of the DNA adduct/DNA polymerase pairs in both binary and ternary complexes. Two isomeric etheno guanine adducts differ considerably in their interactions with DNA polymerases, explaining the base preferences. Further, even when several DNA polymerases cause the same mispairs with a single DNA adduct, the structural bases for this can differ considerably.
PhnJ – A Novel Radical SAM Enzyme from the C-P Lyase Complex
Siddesh S. Kamat1 and Frank M. Raushel2
1The Skaggs Institute for Chemical Biology, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, USA
2Department of Chemistry, Texas A&M University, College Station, USA
PhnJ from the C–P lyase complex catalyzes the cleavage of the carbon–phosphorus bond in ribose-1-phosphonate-5-phosphate (PRPn) to produce methane and ribose-1,2-cyclic-phosphate-5-phosphate (PRcP). This protein is a novel radical SAM enzyme that uses glycyl and thiyl radicals as reactive intermediates in the proposed reaction mechanism. The overall reaction is initiated with the reductive cleavage of S-adenosylmethionine (SAM) by a reduced [4Fe–4S]1+-cluster to form an Ado-CH2∙ radical intermediate. This intermediate abstracts the proR hydrogen from Gly-32 of PhnJ to form Ado-CH3 and a glycyl radical. In the next step, there is hydrogen atom transfer from Cys-272 to the Gly-32 radical to generate a thiyl radical. The thiyl radical attacks the phosphorus center of the substrate, PRPn, to form a transient thiophosphonate radical intermediate. This intermediate collapses via homolytic C–P bond cleavage and hydrogen atom transfer from the proS hydrogen of Gly-32 to produce a thiophosphate intermediate, methane, and a radical intermediate at Gly-32. The final product, PRcP, is formed by nucleophilic attack of the C2-hydroxyl on the transient thiophosphate intermediate. This reaction regenerates the free thiol group of Cys-272. After hydrogen atom transfer from Cys-272 to the Gly-32 radical, the entire process is repeated with another substrate molecule without the use of another molecule of SAM or involvement from the [4Fe–4S]-cluster again.
The Accidential Assignment of Function in the Tautomerase Superfamily
Jamison P. Huddleston, William H. Johnson, Gottfired K. Schroeder and Christian P. Whitman
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, USA
Cg10062 from Corynebacterium glutamicum is a tautomerase superfamily member with the characteristic β−α−β fold and catalytic Pro-1. It is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue with high sequence similarity (53%) that includes the six critical active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114). However, Cg10062 is a poor cis-CaaD: it has much lower catalytic efficiency and lacks isomer specificity. Two acetylene compounds (propiolate and 2-butynoate) and an allene (2,3-butadienote) were investigated as potential substrates for Cg10062. Cg10062 is a hydratase/decarboxylase using propiolate and cis-3-chloro- and 3-bromoacrylates, where malonate semialdehyde is the product of hydration and acetaldehyde is the product of decarboxylation. The two activities occur consecutively using the initial substrate. In contrast, 2-butynoate and 2,3-butadienote only undergo a hydration reaction with Cg10062 to afford acetoacetate. cis-CaaD does not function as a hydratase/decarboxylase using any of these substrates, yielding only the products of hydration. Cg10062 proceeds by direct hydration or covalent catalysis (using Pro-1) depending on the substrate. Direct hydration yields the hydration products and covalent catalysis yields the hydration and decarboxylation products. Cg10062 mutants shift the reaction toward one or the other mechanism. The observation that propiolate is the best substrate suggests that Cg10062 could be a hydratase/decarboxylase in a pathway that transforms an unknown acetylene compound to acetaldehyde via propiolate. The bifunctional activity of Cg10062 might also have implications for the evolution of the dehalogenase and decarboxylase activities in the tautomerase superfamily.
The Catalytic Reaction Mechanism of Drosophilid Alcohol Dehydrogenases
Imin Wushur, Ingebrigt Sylte and Jan-Olof Winberg
Department of Medical Biology, Faculty of Health Sciences, UiT-The Arctic University of Norway, Tromsø, Norway
The present review describes the current knowledge about the reaction mechanism of drosophilid alcohol dehydrogenases (DADH), a member of the short chain dehydrogenase/reductase (SDR) superfamily. Included is the binding order of the substrates to the enzyme, rate limiting steps, stereochemistry of the reaction, active site topology, role of important amino acids and water molecules in the reaction and pH dependence of kinetic coefficients. We focus on the contribution from steady state kinetics where alternative substrates, dead end and product inhibitors, isotopes and mutated DADHs have been used as well as on the contributions from X-ray crystallography, NMR and theoretical calculations. Furthermore, we also raise some open questions in order to fully understand the reaction mechanism of this enzyme.
Some Novel Feature of Glutathion Transferase from Juvenile Catfish (Clarias gariepinus) Exposed to Lindane-contaminated Water
1, , , 3
1Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
2Department of Biological Sciences, College of Science, Engineering and Technology, Osun State University, Osogbo, Nigeria
3Department of Medical Biochemistry, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria
Catfish are hardy in nature and it is not known whether the presence of efficient detoxication enzymes is partly responsible for this trait. To investigate this, we have assessed induction of glutathione transferase (GST) in 10-week-old juvenile catfish (Clarias gariepinus) exposed to graded concentrations of lindane, an organochlorine insecticide, and characterised the purified enzyme from groups having the highest and statistically significant induction. Some of the unique properties observed for the purified enzyme are a high Km (1.72±0.21 mM) for the electrophilic model substrate, 1-chloro-2,4-dinitrobenzene (CDNB) and a very low catalytic rate (Vmax=0.130±0.010 units/mg protein). The kcat/Km being 55.4±0.2 M−1 s−1. The enzyme is present in high concentration in the organism, the main isoform accounts for about 5.6% of the total soluble protein, probably to compensate for the observed kinetic imperfection. Since these properties are generally not known for a detoxication enzyme, we suggest that they may form part of the organism׳s own adaptation to its polluted environment.