Biochemical kinetics and reaction mechanisms

Biochemical reactions carried out by enzymes are fundamental to the metabolic processes of catabolism, and anabolism. Similarly the binding events and signal modifications that are carried out by signaling and receptors proteins are important in the control functions of an organism. Studies of the mechanisms of these important biochemical agents allows insights into how an organisms functions at the molecular level.

The kinetics of biochemical reactions involves the study of rates of chemical processes involved in many processes. Measurements of the rates of reactions under different experimental conditions (for instance pH, solvent, concentration and temperature) allow the construction of models, using software tools like Pro-K II, that describe the characteristics of a biochemical reaction. This model provides insights into the reaction mechanisms involved in the reaction. The most important mechanistic reactions can be broadly classed as binding events, and enzymatic catalysis.

Binding events, such a protein-ligand binding and release, are fundamental to all biochemistry, from signaling pathways to binding of reactants in enzymatic reactions. The use of stopped-flow spectrophotometer are particularly powerful tools to study the kinetics of binding reactions, using fluorescence and other optical probes. This can provide information about the mechanisms of binding, and the energies involved.

Enzymatic reactions catalyze the conversion of metabolites and are the agents that carry out the large variety of specific chemical reactions in biology. As with all chemical kinetics, understanding of the number and rate of reactions, and building of reactions models around this information can provide profound insights into the mechanism of actions at the chemical level for enzymatic reactions.

Stopped-flow spectrophotometers like the SX20 allow the following of reaction kinetics, initiated by the mixing of two (or more) reactants, from the millisecond time range onwards, using changes in various optical probes, like fluorescence, fluorescence anisotropy, absorbance and circular dichroism. This allows a very diverse range of mechanism of many different types of biochemical reactions to be studied in great depth. For a more in depth explanation of the stopped-flow method please read the tutorial.

Laser flash photolysis using the LKS.60 allows the reactions occurring in the nanosecond time range to be studied. Reactions are initiated by a very brief pulse of laser light. Then data is collected using a number of spectrometric techniques. This allows ultrafast reactions processes to be studied. For a more in depth explanation of the laser flash photolysis method please read the tutorial.

Relevant Binding Mechanisms References

Listed below are 5 selected recent references of studies of various binding and macromolecular interaction mechanisms using APL stopped-flow and laser flash photolysis systems. A complete searchable database with all references can be accessed by logging into the APL members area.

Authors	Title	Year	Keywords	Journal/Proceedings
Oleg V. Moskvin, Samuel Kaplan, Marie-Alda Gilles-Gonzalez, and Mark Gomelsky	Novel Heme-based Oxygen Sensor with a Revealing Evolutionary History [Abstract]   [URL]	2007	Signal transduction, Oxygen Sensor, hemeprotein	J BIOL CHEM, 2007, Vol 282, Iss 39, pp 28740-28748
Abstract: To monitor fluctuations in oxygen concentration, cells use sensory proteins often containing heme cofactors. Here, we identify a new class of heme-binding oxygen sensors, reveal their unusual phylogenetic origin, and propose a sensing mode of a member of this class. We show that heme is bound noncovalently to the central region of AppA, an oxygen and light sensor from Rhodobacter sphaeroides. The addition of oxygen to ferrous AppA discoordinated the heme, and subsequent oxygen removal fully restored the heme coordination. In vitro, the extent of heme discoordination increased gradually with the rise in oxygen levels over a broad concentration range. This response correlated well with the gradual decrease in transcription of photosynthesis genes regulated by AppA and its partner repressor PpsR. We conclude that the AppA-PpsR regulatory system functions as an oxygen-dependent transcriptional rheostat. We identified a new domain embedded in the central region of AppA and designated it SCHIC for sensor containing heme instead of cobalamin. A phylogenetic analysis revealed that SCHIC domain proteins form a distinct cluster within a superfamily that includes vitamin B12-binding proteins and other proteins that may bind other kinds of tetrapyrroles.
Celestine N Chi, Lisa Elfstrom, Yao Shi, Tord Snall, Ake Engstrom, Per Jemth	Reassessing a sparse energetic network within a single protein domain [Abstract]   [URL]	2008	allostery, coupling energy, dynamics, energetic network of residues, PDZ domain	PNAS 2008 vol. 105 no. 12 pp 4679-4684
Abstract: Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295–299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.
Dina Grohmann, Dr., Valentina Corradi, Dr., Mira Elbasyouny , Annika Baude , Florian Horenkamp, Sandra D. Laufer , Fabrizio Manetti, Dr. , Maurizio Botta, Prof. , Tobias Restle, Prof.	Small Molecule Inhibitors Targeting HIV-1 Reverse Transcriptase Dimerization [Abstract]   [URL]	2008	antiviral agents, dimerization, drug design , HIV reverse transcriptase	ChemBioChem Volume 9 Issue 6, Pages 916 - 922
Abstract: The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t1/2 of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.
Hannah S. Timsa and Jonathan Widom	Stopped-flow fluorescence resonance energy transfer for analysis of nucleosome dynamics [Abstract]   [URL]	2007	Chromatin; Histones; LexA protein; DNA; Nucleosome reconstitution; FRET; Kinetics; Cy-dye labeling; Conformational dynamics; Site exposure	Methods Volume 41, Issue 3, March 2007, Pages 296-303
Abstract: Macromolecular assemblies and machines undergo large-scale conformational changes as essential features of their normal function. Modern stopped-flow instrumentation and biotechnology combine to provide a powerful tool for characterizing the rates and natures of these conformational changes. Standard commercially available instruments provide extraordinary sensitivity and speed, allowing analysis of millisecond or longer timescale processes, with concentrations as low as a few nanomolar and volumes of just a few hundred microliters. One can now place specific dyes anywhere desired on a nucleic acid, and often on a protein as well. This ability allows the use of fluorescence resonance energy transfer experiments for detailed conformational analyses, even as the system is evolving rapidly over time following the initiation of a reaction. This approach is ideally suited for analysis of intrinsic properties of chromatin and of the machines that control chromatin assembly, disassembly, and function.
Yannick H. Ouellet, Richard Daigle, Patrick Lagüe, David Dantsker, Mario Milani, Martino Bolognesi, Joel M. Friedman, and Michel Guertin	Ligand Binding to Truncated Hemoglobin N from Mycobacterium tuberculosis Is Strongly Modulated by the Interplay between the Distal Heme Pocket Residues and Internal Water [Abstract]   [URL]	2008	Nitric oxide, laser flash photolysis, hemoglobin, binding	J. Biol. Chem., Vol. 283, Issue 40, 27270-27278, October 3.
Abstract: The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (Formula {approx} 745 x 106 M-1·s-1), which is ~15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 x 109 M-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (~1.49 x 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).

Relevant Enzyme Mechanisms References

Listed below are 5 selected recent references of studies a number of enzyme kinetic mechanisms using APL stopped-flow and laser flash photolysis systems. A complete searchable database with all references can be accessed by logging into the APL members area.

Authors	Title	Year	Keywords	Journal/Proceedings
Emile Bol, Nicolette J. Broers, and Wilfred R. Hagen	A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus [Abstract]   [URL]	2008	Tungsten, Formaldehyde oxidoreductase, Pyrococcus furiosus, Pre-steady-state kinetics, Steady-state kinetics	J BIOL INORG CHEM, 2008, Vol 13, Iss 1, pp 75-84
Abstract: Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM-1 cm-1) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s-1, kobs2 = 1.9 s-1) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 × 10-2 s-1, kobs4 = 2.18 × 10-2 s-1). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.
Hothi, P.; Lee, M.; Cullis, P. M.; Leys, D.; Scrutton, N. S.	Catalysis by the Isolated Tryptophan Tryptophylquinone-Containing Subunit of Aromatic Amine Dehydrogenase Is Distinct from Native Enzyme and Synthetic Model Compounds and Allows Further Probing of TTQ Mechanism [Abstract]   [URL]	2008	benzylamines, Aromatic Amine Dehydrogenase, TTQ Mechanism	BIOCHEMISTRY-USA, 2008, Vol 47, Iss 1, pp 183-194
Abstract: Para-substituted benzylamines are poor reactivity probes for structure-reactivity studies with TTQ-dependent aromatic amine dehydrogenase (AADH). In this study, we combine kinetic isotope effects (KIEs) with structure-reactivity studies to show that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated TTQ-containing subunit of AADH. Contrary to the TTQ-containing subunit of methylamine dehydrogenase (MADH), which is catalytically inactive, the small subunit of AADH catalyzes the oxidative deamination of a variety of amine substrates. Observed rate constants are second order with respect to substrate and inhibitor (phenylhydrazine) concentration. Kinetic studies with para-substituted benzylamines and their dideuterated counterparts reveal KIEs (>6) larger than those observed with native AADH (KIEs ~ unity). This is attributed to formation of the benzylamine-derived iminoquinone requiring structural rearrangement of the benzyl side chain in the active site of the native enzyme. This structural reorganization requires motions from the side chains of adjacent residues (which are absent in the isolated small subunit). The position of Phe97 in particular is responsible for the conformational gating (and hence deflated KIEs) observed with para-substituted benzylamines in the native enzyme. Hammett plots for the small subunit exhibit a strong correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenethylamines, unlike native AADH for which a poor correlation is observed. TTQ reduction in the isolated subunit is enhanced by electron withdrawing substituents, contrary to structure-reactivity studies reported for synthetic TTQ model compounds in which rate constants are enhanced by electron donating substituents. We infer that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated small subunit. This is attributed to the absence of structural rearrangement prior to H-transfer that limits the rate of TTQ reduction by para-substituted benzylamines in native enzyme.
Shiva Bhowmik, Geoff P. Horsman, Jeffrey T. Bolin, and Lindsay D. Eltis	The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation: LARGE 3-SUBSTITUENTS PREVENT TAUTOMERIZATION [Abstract]   [URL]	2007	polychlorinated biphenyls, PCB, Bph, BphD, catalysis	J BIOL CHEM, 2007, Vol 282
Abstract: The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A·3-Cl HOPDA and S112A·3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A·HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 Å from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A·3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.
William C. Cooper, Yi Jin, and Trevor M. Penning	Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate: THE EXAMPLE OF RAT LIVER 3α-HSD (AKR1C9) [Abstract]   [URL]	2007	Hydroxysteroid dehydrogenases, steroid biosynthesis, enzyme kinetics	J BIOL CHEM, 2007, Vol 282, Iss 46, pp 33484-33493
Abstract: Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3α-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP+ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP+ controlled kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated kcat, whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.
Derren J. Heyes, Michiyo Sakuma, and Nigel S. Scrutton	Laser Excitation Studies of the Product Release Steps in the Catalytic Cycle of the Light-driven Enzyme, Protochlorophyllide Oxidoreductase [Abstract]   [URL]	2007	Protochlorophyllide Oxidoreductase, Light-driven Enzyme, Laser Excitation, enzyme catalysis	J BIOL CHEM, 2007, Vol 282, Iss 44, pp 32015-32020
Abstract: The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s–1. The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of ~72 s–1. At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s–1. The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.