Mechanism Of Substrate Activation Research Articles

Distinguishing between Linear and Non-Linear (Cooperative) Substrate Activation Mechanisms in the Sonogashira Reaction under “Ligand-Free” and “Copper-Free” Conditions

The results are presented on the comparative studies of the differential selectivity patterns in the Sonogashira reaction with a pair of competing aryl acetylenes in the “ligand-free” and “copper-free” conditions when varying the nature and concentration of aryl halides and base. The revealed sensitivity of the differential selectivity of competing aryl acetylenes to aryl halide nature unambiguously indicated that the substrates were activated through linear mechanism from kinetic view. An absence of any influence of the nature and concentration of the base on the differential selectivity of competing aryl acetylenes indicated the irreversible character of the step of their activation.

To the Understanding of Catalysis by D-Amino Acid Transaminases: A Case Study of the Enzyme from Aminobacterium colombiense

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from Bacillus subtilis. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium Aminobacterium colombiense with a substrate binding mode different from that for the transaminase from B. subtilis. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (R)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism.

Iodine-Iodine Cooperation Enables Metal-Free C-N Bond-Forming Electrocatalysis via Isolable Iodanyl Radicals.

Small molecule redox mediators convey interfacial electron transfer events into bulk solution and can enable diverse substrate activation mechanisms in synthetic electrocatalysis. Here, we report that 1,2-diiodo-4,5-dimethoxybenzene is an efficient electrocatalyst for C-H/E-H coupling that operates at as low as 0.5 mol % catalyst loading. Spectroscopic, crystallographic, and computational results indicate a critical role for a three-electron I-I bonding interaction in stabilizing an iodanyl radical intermediate (i.e., formally I(II) species). As a result, the optimized catalyst operates at more than 100 mV lower potential than the related monoiodide catalyst 4-iodoanisole, which results in improved product yield, higher Faradaic efficiency, and expanded substrate scope. The isolated iodanyl radical is chemically competent in C-N bond formation. These results represent the first examples of substrate functionalization at a well-defined I(II) derivative and bona fide iodanyl radical catalysis and demonstrate one-electron pathways as a mechanistic alternative to canonical two-electron hypervalent iodine mechanisms. The observation establishes I-I redox cooperation as a new design concept for the development of metal-free redox mediators.

Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism.

β-Ureidopropionase (βUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like βUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human βUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N-carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer-dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly βUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer-dimer interface, effectively positioning the catalytically important Glu207 in the active site.

Terminal Olefin Profiles and Phylogenetic Analyses of Olefin Synthases of Diverse Cyanobacterial Species.

Cyanobacteria can synthesize alkanes and alkenes, which are considered to be infrastructure-compatible biofuels. In terms of physiological function, cyanobacterial hydrocarbons are thought to be essential for membrane flexibility for cell division, size, and growth. The genetic basis for the biosynthesis of terminal olefins (1-alkenes) is a modular type I polyketide synthase (PKS) termed olefin synthase (Ols). The modular architectures of Ols and structural characteristics of alkenes have been investigated only in a few species of the small percentage (approximately 10%) of cyanobacteria that harbor putative Ols pathways. In this study, investigations of the domains, modular architectures, and phylogenies of Ols in 28 cyanobacterial strains suggested distinctive pathway evolution. Structural feature analyses revealed 1-alkenes with three carbon chain lengths (C15, C17, and C19). In addition, the total cellular fatty acid profile revealed the diversity of the carbon chain lengths, while the fatty acid feeding assay indicated substrate carbon chain length specificity of cyanobacterial Ols enzymes. Finally, in silico analyses suggested that the N terminus of the modular Ols enzyme exhibited characteristics typical of a fatty acyl-adenylate ligase (FAAL), suggesting a mechanism of fatty acid activation via the formation of acyl-adenylates. Our results shed new light on the diversity of cyanobacterial terminal olefins and a mechanism for substrate activation in the biosynthesis of these olefins.IMPORTANCE Cyanobacterial terminal olefins are hydrocarbons with promising applications as advanced biofuels. Despite the basic understanding of the genetic basis of olefin biosynthesis, the structural diversity and phylogeny of the key modular olefin synthase (Ols) have been poorly explored. An overview of the chemical structural traits of terminal olefins in cyanobacteria is provided in this study. In addition, we demonstrated by in vivo fatty acid feeding assays that cyanobacterial Ols enzymes might exhibit substrate carbon chain length specificity. Furthermore, by performing bioinformatic analyses, we observed that the substrate activation domain of Ols exhibited features typical of a fatty acyl-adenylate ligase (FAAL), which activates fatty acids by converting them to fatty acyl-adenylates. Our results provide further insight into the chemical structures of terminal olefins and further elucidate the mechanism of substrate activation for terminal olefin biosynthesis in cyanobacteria.

Catalytic Fate of Two Copper Complexes towards Phenoxazinone Synthase and Catechol Dioxygenase Activity

AbstractIn this present work, we report the synthesis and structural characterization of two copper(II) complexes, [Cu(bpy)Cl2] (1) & [Cu(μ‐Cl)(phen)Cl]2 (2) [bpy=2,2′‐bipyridine; phen=1,10‐phenanthroline]. We have also studied their catalytic fate towards phenoxazinone synthase and catechol dioxygenase activity. X‐ray structural analyses revealed that 1 & 2 crystallize in triclinic & monoclinic system with P 1 and Cc space group respectively. The copper complexes catalyse the oxidative coupling of 2‐amino phenol (2‐AP) to aminophenoxazin‐3‐one with significant turn over number, kcat(h−1)=2.08×103 & 2.16×103 for 1 & 2 resectively. During investigation of catechol dioxygenase activity, stoichiometric addition of 1 & 2 to 3,5‐di‐tert‐butylcatechol (DTBC) in acetonitrile produce in situ catecholate‐to‐Cu(II) absorption bands at 812 and 821 nm respectively. The in situ Cu(II)‐catecholate species for both 1 & 2 react with molecular oxygen at the rate, kobs: 7.95×10−4 and 1.30×10−3 min−1 respectively and produce intradiol cleavage products in exclusive amount. Minor amount of benzoquinone is also found in solution. Intradiol products are found as major product in solution and accounts in favour of substrate activation mechanism.

Reversal of Enantioselectivity Approach to BINOLs via Single and Dual 2-Naphthol Activation Modes.

A mechanism-driven enantiodivergent approach to chiral 1,1'-bi-2-naphthols via catalytic asymmetric oxidative coupling of 2-naphthol derivatives is described for the first time. By utilizing 2-naphthol derivatives with low oxidation potential, the substrates were activated by either chiral mononuclear or dinuclear vanadium(V) catalyst to promote distinctively different asymmetric reaction pathways: single versus dual substrate activation mechanisms.

Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation

The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

Catalytic promiscuity of an iron(II)–phenanthroline complex

A mononuclear iron(II) complex, [Fe(phen)3]Cl2 (1) (phen =1,10‐phenanthroline), has been synthesized in crystalline phase and characterized using various spectroscopic techniques including single crystal X‐ray diffraction. Crystal structure analysis revealed that 1 crystallizes in a monoclinic system with C2/m space group. Complex 1 acts as a functional model for a biomimetic catalyst promoting the aerobic oxidation of 3,5‐di‐tert‐butylcatechol (3,5‐DTBC) through radical pathways with a significant turnover number (kcat =3.55 × 103 h−1) and exhibits catechol dioxygenase activity towards the same 3,5‐DTBC substrate at room temperature in oxygen‐saturated ethanol medium. The existence of an isobestic point at 610 nm from spectrophotometric data indicates the presence of Fe3+ −3,5‐DTBC adduct favouring an enzyme–substrate binding phenomenon. Upon stoichiometric addition of 3,5‐DTBC pretreated with two equivalents of triethylamine to the iron complex, two catecholate‐to‐iron(III) ligand‐to‐metal charge transfer bands (575 and 721 nm) are observed and the in situ generated catecholate intermediate reacts with dioxygen (kobs =9.89 × 10−4 min−1) in ethanol medium to afford exclusively intradiol cleavage products along with a small amount of benzoquinone, and a small amount of extradiol cleavage products, which provide substantial evidence for a substrate activation mechanism. Copyright © 2016 John Wiley & Sons, Ltd.

Di- and Trinuclear Mixed-Valence Copper Amidinate Complexes from Reduction of Iodine.

Molecular examples of mixed-valence copper complexes through chemical oxidation are rare but invoked in the mechanism of substrate activation, especially oxygen, in copper-containing enzymes. To examine the cooperative chemistry between two metals in close proximity to each other we began studying the reactivity of a dinuclear Cu(I) amidinate complex. The reaction of [(2,6-Me2C6H3N)2C(H)]2Cu2, 1, with I2 in tetrahydrofuran (THF), CH3CN, and toluene affords three new mixed-valence copper complexes [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I3)(THF)2, 2, [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I) (NCMe)2, 3, and [(2,6-Me2C6H3N)2C(H)]3Cu3(μ3-I)2, 4, respectively. The first two compounds were characterized by UV-vis and electron paramagnetic resonance spectroscopies, and their molecular structure was determined by X-ray crystallography. Both di- and trinuclear mixed-valence intermediates were characterized for the reaction of compound 1 to compound 4, and the molecular structure of 4 was determined by X-ray crystallography. The electronic structure of each of these complexes was also investigated using density functional theory.

Abstract 2158: PDK1-dependent activation of RSK is an absolute requirement for PI3K oncogenic activity in the thyroid gland

Abstract Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a key role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC). Accordingly, conditional deletion of Pten in the mouse thyroid epithelium induces constitutive activation of the PI3K/AKT pathway, and causes thyroid hyperplasia at birth that progresses to invasive and metastatic follicular carcinoma. This phenotype is dramatically accelerated by simultaneous deletion of Trp53, leading to the development of ATC, or activation of Kras, leading to poorly differentiated carcinomas. The PDK1 kinase plays a central role in the PI3K signaling cascade, acting as a master regulator of PI3K downstream effectors belonging to the AGC kinases family, such as AKT and S6K1. Additional AGC kinases are controlled by PDK1 independent of PI3K activity. PDK1 possesses a substrate docking site, the “PIF pocket”, required for the phosphorylation of most AGC kinases (including S6K, PKC, SGK, and RSK) but not of AKT. The L155E PDK1 mutant disrupts the PIF pocket and impairs AGC kinase activation, without any effect on AKT activation. Thanks to this differential mechanism of substrate activation, we are defining the relative contribution of AKT and other PDK1 targets to the neoplastic transformation process in Pten-/- thyrocytes. Surprisingly, in vivo disruption of the PIF-pocket completely abrogates neoplastic transformation of Pten-/- thyroids, even in the face of AKT constitutive activation. This finding supports the hypothesis that the linear PI3K-AKT pathway is not sufficient to drive thyroid transformation and that concomitant activation of additional PDK1-dependent pathways is absolutely required. We now show that RSK family members (RSK1-3) are essential components of this PDK1-dependent accessory pathway. Remarkably, RSK3 levels in the thyroid are increased in the absence of Pten, and RSK activity in thyroid cells is abrogated when PDK1 PIF-pocket is mutated. Genetic and pharmacological inhibition of RSK drastically impairs [Pten,Trp53]-/- ATC cell proliferation both in vitro and in vivo, in syngeneic allograft models. Taken together, our data identify a new signaling cascade that is essential for neoplastic transformation of thyroid epithelial cells, and thus a new critical candidate for drug development. Citation Format: Arturo Orlacchio, Antonio Di Cristofano. PDK1-dependent activation of RSK is an absolute requirement for PI3K oncogenic activity in the thyroid gland. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2158. doi:10.1158/1538-7445.AM2015-2158

Bond-weakening catalysis: conjugate aminations enabled by the soft homolysis of strong N-H bonds.

The ability of redox-active metal centers to weaken the bonds in associated ligands is well precedented, but has rarely been utilized as a mechanism of substrate activation in catalysis. Here we describe a catalytic bond-weakening protocol for conjugate amination wherein the strong N-H bonds in N-aryl amides (N-H bond dissociation free energies ∼100 kcal/mol) are destabilized by ∼33 kcal/mol upon by coordination to a reducing titanocene complex, enabling their abstraction by the weak H-atom acceptor TEMPO through a proton-coupled electron transfer process. Significantly, this soft homolysis mechanism provides a method to generate closed-shell, metalated nucleophiles under neutral conditions in the absence of a Brønsted base.

Metabolism of Halogenated Alkanes by Cytochrome P450 enzymes. Aerobic Oxidation versus Anaerobic Reduction

The cytochromes P450 are a large class of heme-containing enzymes that catalyze a broad range of chemical reactions in biosystems, mainly through oxygen-atom transfer to substrates. A relatively unknown reaction catalyzed by the P450s, but very important for human health, is the activation of halogenated substrates, which may lead to toxicity problems. However, its catalytic mechanism is currently unknown and, therefore, we performed a detailed computational study. To gain insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and reductive P450-mediated activation of tetra- and trichloromethane as halogenated models with density functional theory (DFT) methods. We propose an oxidative halosylation mechanism for CCl4 under aerobic conditions by Compound I of P450, which follows the typical Groves-type rebound mechanism. By contrast, the metabolism of CHCl3 occurs preferentially via an initial hydrogen-atom abstraction rather than halosylation. Kinetic isotope effect studies should, therefore, be able to distinguish the mechanistic pathways of CCl4 versus CHCl3 . We find a novel mechanism that is different from the well accepted P450 substrate activation mechanisms reported previously. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized using Marcus theory equations, which reproduce experimental product distributions.

Catalytic Ketyl-Olefin Cyclizations Enabled by Proton-Coupled Electron Transfer

Concerted proton-coupled electron transfer is a key mechanism of substrate activation in biological redox catalysis. However, its applications in organic synthesis remain largely unexplored. Herein, we report the development of a new catalytic protocol for ketyl-olefin coupling and present evidence to support concerted proton-coupled electron transfer being the operative mechanism of ketyl formation. Notably, reaction outcomes were correctly predicted by a simple thermodynamic formalism relating the oxidation potentials and pKa values of specific Brønsted acid/reductant combinations to their capacity to act jointly as a formal hydrogen atom donor.

Iron(III) Complexes with Meridional Ligands as Functional Models of Intradiol-Cleaving Catechol Dioxygenases

Six dichloroiron(III) complexes of 1,3-bis(2'-arylimino)isoindoline (BAIH) with various N-donor aryl groups have been characterized by spectroscopy (infrared, UV-vis), electrochemistry (cyclic voltammetry), microanalysis, and in two cases X-ray crystallography. The structurally characterized Fe(III)Cl(2)(L(n)) complexes (n = 3, L(3) = 1,3-bis(2'-thiazolylimino)isoindoline and n = 5, L(5) = 1,3-bis(4-methyl-2'-piridylimino)isoindoline) are five-coordinate, trigonal bipyramidal with the isoindoline ligands occupying the two axial and one equatorial positions meridionally. These compounds served as precursors for catechol dioxygenase models that were formed in solution upon addition of 3,5-di-tert-butylcatechol (H(2)DBC) and excess triethylamine. These adducts react with dioxygen in N,N-dimethylformamide, and the analysis of the products by chromatography and mass spectrometry showed high intradiol over extradiol selectivity (the intradiol/extradiol product ratios varied between 46.5 and 6.5). Kinetic measurements were performed by following the change in the intensity of the catecholate to iron ligand-to-metal charge transfer (LMCT) band, the energy of which is influenced by the isoindolinate-ligand (827-960 nm). In combination with electrochemical investigations the kinetic studies revealed an inverse trend between reaction rates and oxidation potentials associated with the coordinated DBC(2-). On the basis of these results, a substrate activation mechanism is suggested for this system in which the geometry of the peroxide-bridged intermediate may be of key importance in regioselectivity.

ChemInform Abstract: Noncovalent Organocatalysis Based on Hydrogen Bonding: Elucidation of Reaction Paths by Computational Methods

AbstractReview: 64 refs.

Iron(III) Complexes of Tripodal Monophenolate Ligands as Models for Non-Heme Catechol Dioxygenase Enzymes: Correlation of Dioxygenase Activity with Ligand Stereoelectronic Properties

The iron(III) complexes [Fe(L)Cl(2)] 1-6 of the tripodal monophenolate ligands N,N-bis(2-pyridylmethyl)-N'-(2-hydroxybenzyl)amine H(L1), [(1-methylimidazol-2-ylmethyl)-(pyrid-2-ylmethyl)aminomethyl]-phenol H(L2), 2,4-dimethyl-6-[(1-methylimidazol-2-ylmethyl)(pyrid-2-ylmethyl)aminomethyl]phenol H(L3), N,N-dimethyl-N'-(pyrid-2-ylmethyl)-N'-(2-hydroxybenzyl)ethylenediamine H(L4), N,N-dimethyl-N'-(1-methylimidazol-2-ylmethyl)-N'-(2-hydroxybenzyl)ethylenediamine H(L5), and N,N-dimethyl-N'-(1-methylimidazol-2-ylmethyl)-N'-(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine H(L6) have been isolated and studied as structural and functional models for the intradiol-cleaving catechol dioxygenase enzymes. The complexes have been characterized using elemental analysis, electrospray ionization mass spectrometry, and absorption spectral and electrochemical methods. The single crystal X-ray structures of [Fe(L3)Cl(2)] 3 and [Fe(L6)Cl(2)] 6 have been successfully determined, and the rhombically distorted octahedral coordination geometry around iron(III) in them are constituted by the phenolate oxygen and pyridyl/-NMe(2), and N-methylimidazolyl and tertiary amine nitrogens of the tripodal tetradentate ligands H(L3)/H(L6) and two cis-coordinated chloride ions. The sterically demanding -NMe(2) group as in 6 imposes an Fe-O-C bond angle (139.8 degrees) and Fe-O bond length (1.852 A), which are very close to those (Fe-O-C, 133, 148 degrees; Fe-O(tyrosinate), 1.81, 1.91 A) of 3,4-PCD enzymes. The Fe-O-C bond angle observed for 6 is higher than that for 3 (125.1 degrees), and the Fe-O(phenolate) bond distance in 6 is shorter than that in 3 (1.905 A). In methanol solution all the complexes exhibit two phenolate-to-Fe(III) ligand-to-metal charge transfer (LMCT) bands in the ranges 536-622 and 329-339 nm. Further, when 3,5-di-tert-butylcatechol (H(2)DBC) pretreated with two moles of Et(3)N is added to 1-6, two new intense DBC(2-)-to-iron(III) LMCT bands (466-489, 676-758 nm) are observed, which are similar to those observed for 3,4-PCD enzyme-substrate complex. All the complexes elicit oxidative intradiol cleavage of H(2)DBC in the presence of O(2). Interestingly, among the present complexes, 3 containing coordinated N-methylimidazolyl nitrogen shows the highest rate of intradiol cleavage, which correlates with the highest energy of DBC(2-)-to-iron(III) LMCT band and the most negative DBSQ/DBC(2-) redox potential. Also, the catecholate adducts of complexes 4 and 5, both containing a -NMe(2) donor group, react faster and produce higher amounts of intradiol cleavage products (4: 55.3; 5, 50.6%) than the analogous complexes 1 (43.2%) and 2 (32.7%), both containing a pyridyl nitrogen donor, which is consistent with the more negative DBSQ/DBC(2-) redox potentials for 4 and 5. The increase in rate of catechol dioxygenation with increase in the DBC(2-)-to-iron(III) LMCT band energy and decrease in DBSQ/DBC(2-) redox potential is illustrated by invoking a facile alpha-electron transfer from iron(III) to catecholate-bound molecular oxygen in the substrate activation mechanism proposed for the intradiol-cleaving catechol dioxygenases. Also, when the substituents on the phenolate arm are varied to tune the Lewis acidity of iron(III) center, the reaction rate decreases with decrease in Lewis acidity and, interestingly, extradiol cleavage is also observed when the Lewis acidity is decreased further by incorporating a 3,5-dimethylphenolate arm as in 6.

Allosteric activation of pyruvate decarboxylases. A never-ending story?

The allosteric substrate activation of pyruvate decarboxylases was studied for more than 30 years using varying techniques and ending up in different hypotheses on the molecular mechanism of substrate activation of this enzyme. Now, a number of high-resolution structures of the pyruvate decarboxylase species from Saccharomyces cerevisiae and from Kluyveromyces lactis – both wild type and variants in complex with covalently bound substrate or substrate surrogates – provided for the first time structural insights to decipher the mechanism of allosteric activation by describing the signal transduction pathway from the regulatory to the active site in detail. Here, the mechanistic studies on substrate activation of pyruvate decarboxylases are reviewed from a historical point of view, demonstrating that important parts of the different hypotheses got carried away with our new mechanism drawn from latest results of experiments on activation kinetics, small-angle X-ray solution scattering and X-ray crystal diffraction.

Molecular Mechanism of Allosteric Substrate Activation in a Thiamine Diphosphate-dependent Decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

Kinetic and spectroscopic studies of N694C lipoxygenase: a probe of the substrate activation mechanism of a nonheme ferric enzyme.

Lipoxygenases (LOs) comprise a class of substrate activating mononuclear nonheme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. A commonly proposed mechanism for LO catalysis involves H-atom abstraction by an FeIII-OH- site, best described as a proton coupled electron transfer (PCET) process, followed by direct reaction of O2 with the resulting substrate radical to yield product. An alternative mechanism that has also been discussed involves the abstraction of a proton from the substrate by the FeIII-OH leading to a sigma-organoiron intermediate, where the subsequent sigma bond insertion of dioxygen into the C-Fe bond completes the reaction. H-atom abstraction is favored by a high E(o) of the FeII/FeIII couple and high pK(a) of water bound to the ferrous state, while an organoiron mechanism would be favored by a low E(o) (to keep the site oxidized) and a high pK(a) of water bound to the ferric state (to deprotonate the substrate). A first coordination sphere mutant of soybean LO (N694C) has been prepared and characterized by near-infrared circular dichroism (CD) and variable-temperature, variable-field (VTVH) magnetic circular dichroism (MCD) spectroscopies (FeII site), as well as UV/vis absorption, UV/vis CD, and electron paramagnetic resonance (EPR) spectroscopies (FeIII site). These studies suggest that N694C has a lowered E degrees of the FeII/FeIII couple and a raised pKa of water bound to the ferric site relative to wild type soybean lipoxygenase-1 (WT sLO-1) which would favor the organoiron mechanism. However, the observation in N694C of a significant deuterium isotope effect, anaerobic reduction of iron by substrate, and a substantial decrease in k(cat) (approximately 3000-fold) support H-atom abstraction as the relevant substrate-activation mechanism in sLO-1.