Active-site Coupling Research Articles

Engineering of active site coupling to facilitate interatomic charge transfer for bifunctional oxygen electrocatalysis

Interatomic charge transfer emerges as a robust technique for enhancing catalytic efficacy in key energy reactions, specifically the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). Despite its potential, advancing simultaneous improvements in reversible oxygen catalysis pose considerable challenges. Herein, we detailed an active site coupling engineering method to create a CoCu@NSC bifunctional oxygen electrode. In this configuration, OER-active Cu sites were alloyed with ORR-active Co within a N/S-doping carbon nanobox. This arrangement yielded a half-wave potential of 0.924 V for ORR, alongside an overpotential of η10 = 356 mV for OER. The fabricated CoCu@NSC cathode yielded a discharge capacity of 768 mAh/gZn, a peak power density of 295 mW/cm2, and extensive cycling durability. Work function assessments indicated that the chemical integration of Co and Cu components at the atomic level promoted interfacial charge redistribution, effectively optimizing the adsorption-desorption dynamics of intermediates at the active sites, thereby substantially enhancing the catalytic performance for both OER and ORR. Additionally, doping with N/S species enhanced the conductivity of the hollow carbon matrix, facilitating more effective mass and charge transport pathways. These insights illuminate a compelling approach for the design and synthesis of effective bifunctional oxygen electrodes that is pivotal for advanced energy storage advice.

Activation Loop Plasticity and Active Site Coupling in the MAP Kinase, ERK2

Previous studies of the protein kinase, ERK2, using NMR and hydrogen-exchange measurements have shown changes in dynamics accompanying its activation by phosphorylation. However, knowledge about the conformational motions involved is incomplete. Here, we examined ERK2 using long conventional molecular dynamics (MD) simulations starting from crystal structures of phosphorylated (2P) and unphosphorylated (0P) forms. Individual trajectories were run for (5 to 25) μs, totaling 727 μs. The results show unexpected flexibility of the A-loop, with multiple long-lived (>5 μs) conformational states in both 2P- and 0P-ERK2. Differential contact network and principal component analyses reveal coupling between the A-loop fold and active site dynamics, with evidence for conformational selection in the kinase core of 2P-ERK2 but not 0P-ERK2. Simulations of 2P-ERK2 show A-loop states corresponding to restrained dynamics within the N-lobe, including regions around catalytic residues. One A-loop conformer forms lasting interactions with the L16 segment, leading to reduced RMSF and greater compaction in the active site. By contrast, simulations of 0P-ERK2 reveal excursions of A-loop residues away from the C-lobe, leading to greater active site mobility. Thus, the A-loop in ERK2 switches between distinct conformations that reflect coupling with the active site, possibly via the L16 segment. Crystal packing interactions suggest that lattice contacts with the A-loop may restrain its structural variation in X-ray structures of ERK2. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site.

Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation.

GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371–375 holding catalytic residues and in loop 376–401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk. These studies reveal the mechanism of domain rotation and inter-domain communication, providing a molecular framework for the function of all single polypeptide GMPSs and form a solid basis for rational drug design targeting this therapeutically important enzyme.

Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.

Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase

Numerous steady-state kinetic studies have examined the complex catalytic reaction mechanism of the multifunctional enzyme, pyruvate carboxylase (PC). Through initial velocity, product inhibition, isotopic exchange and alternate substrate experiments, early investigators established that PC catalyzes the MgATP-dependent carboxylation of pyruvate by HCO3 (-) through a nonclassical sequential Bi Bi Uni Uni reaction mechanism. This review surveys previous steady-state kinetic investigations of PC and evaluates the proposed hypotheses concerning the overall catalytic mechanism, nonlinear kinetics and active site coupling in the context of recent structural and mutagenic analyses of this multifunctional enzyme. The determination several PC holoenzyme structures have aided in corroborating the proposed molecular mechanisms by which catalysis occurs and established the inextricable link between the dynamic protein motions and complex kinetic mechanisms associated with PC activity. Unexpectedly, the conclusions drawn from these early steady-state kinetic investigations have consistently proven to be in fundamental agreement with our current understanding of PC catalysis, which is a testament to the overarching sophistication of the methods pioneered by Michaelis and Menten and further developed by Northrop, Cleland and others.

Hydrogen production by steam reforming of dimethyl ether over ZnO–Al2O3 bi-functional catalyst

A series of ZnO–Al2O3 catalysts with various ZnO/(ZnO + Al2O3) molar ratios have been developed for hydrogen production by dimethyl ether (DME) steam reforming within microchannel reactor. The catalysts were characterized by N2 adsorption-desorption, X-ray diffraction and temperature programmed desorption of NH3. It was found that the catalytic activity was strongly dependent on the catalyst composition. The overall DME reforming rate was maximized over the catalyst with ZnO/(ZnO + Al2O3) molar ratio of 0.4, and the highest H2 space time yield was 315 mol h−1·kgcat−1 at 460 °C. A bi-functional mechanism involving catalytic active site coupling has been proposed to account for the phenomena observed. An optimized bi-functional DME reforming catalyst should accommodate the acid sites and methanol steam reforming sites with a proper balance to promote DME steam reforming, whereas all undesired reactions should be impeded without sacrificing activity. This work suggests that an appropriate catalyst composition is mandatory for preparing good-performance and inexpensive ZnO–Al2O3 catalysts for the sustainable conversion of DME into H2-rich reformate.

Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis

Catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys in the primary sequence of the dihydrolipoyl S-acetyltransferase (E2) component. Arabidopsis thaliana plastidial E2 (AtplE2-1A-His(6)) was expressed in Escherichia coli. Analysis of recombinant protein by SDS-PAGE revealed a Mr 59,000 band. Supplementation of bacterial culture medium with l-lipoic acid (LA) shifted the band to Mr 57,000. Intact mass determinations using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) revealed the faster migrating E2 species was 189Da larger than the slower migrating form, exactly the difference that would result from addition of a single lipoamide group. Results from systematic MALDI-TOF analysis of Lys-containing tryptic peptides derived from purified recombinant AtplE2-1A indicate that Lys96 is the site of lipoyl-addition. Analysis of Lys96 site-directed mutant proteins showed that they migrated as single species during SDS-PAGE when expressed in either the absence or presence of supplemental LA. Results from both intact and tryptic peptide mass determinations by MALDI-TOF MS confirmed that the mutant proteins were not lipoylated. The A.thaliana plastidial E2 subunit includes a single lipoyl-prosthetic group covalently attached to Lys96. Despite low primary sequence identity with bacterial E2, the plant E2 protein was recognized and modified by E.coli E2 lipoyl-addition system. Results from meta-genomic analysis suggest a β-turn is more important in defining the site for LA addition than a conserved sequence motif.

Catalytic Production of Liquid Fuels from Biomass‐Derived Oxygenated Hydrocarbons: Catalytic Coupling at Multiple Length Scales

The catalytic processing of biomass‐derived feedstocks to liquid fuels and chemical intermediates is complex and expensive. Therefore, conversion processes involving a limited number of reaction, separation, and purification steps are necessary. Coupling of catalytic processes has the potential to lead to the development of new processes, thereby improving the overall economics of biomass conversion. Functional coupling at the molecular scale has the potential to produce novel catalytic materials to replace homogeneous catalysts. Active site coupling of different sites within the same reactor can help reduce operating costs by combining sequential reactions in a single reactor. Chemical reaction coupling of heterogeneous and homogeneous reactions may lead to improvements in overall catalytic performance for liquid phase processes by enhancing surface reactions with liquid phase reactions. Finally, phase coupling leads to improvements in overall yield by improving the equilibrium conversion or by suppressing undesired side reactions.

An ancestral glutamine-dependent NAD + synthetase revealed by poor kinetic synergism

NAD + synthetase catalyzes the formation of NAD + from ATP, nicotinic acid adenine dinucleotide and ammonia. Glutamine-dependent NAD + synthetase obtains ammonia through the hydrolysis of glutamine to glutamate, which takes place in the glutaminase domain. The ammonia is subsequently transported to the synthetase domain through an interdomain ammonia tunnel. NAD + synthetase from the thermophilic bacteria Thermotoga maritima was cloned and expressed. Steady-state kinetics and stoichiometric analysis of product formation revealed an enzyme that is significantly inefficient in the synchronization of the two active sites resulting in wasteful hydrolysis of glutamine and that is not specific for glutamine over ammonia. Phylogenetic analysis of glutamine-dependent NAD + synthetases identifies three main groups remotely related. The T. maritima NAD + synthetase's group is proposed to represent the ancestral group based on the phylogenetic analysis and on the kinetic characterizations. The phylogenetic results nicely correlate also with the degree of catalytic efficiency measured for M. tuberculosis, S. cerevisiae and T. maritima NAD + synthetases. Furthermore, the data here reported in combination with structural data available for glutamine-dependent NAD + synthetase lays the foundation for further investigation on the mechanism of active site coupling in these enzymes.

Regulation of active site coupling in glutamine-dependent NAD+ synthetase

NAD(+) is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD(+) biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD(+) synthetase, which catalyzes the ATP-dependent formation of NAD(+) at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD(+) synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD(+) synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-A intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

Phosphorylation of Serine 264 Impedes Active Site Accessibility in the E1 Component of the Human Pyruvate Dehydrogenase Multienzyme Complex

At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively deficient in pyruvate binding and reductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc by phosphorylation.

A central role for extended polypeptide linkers in establishing the spatial architecture of a pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex is among the largest multifunctional enzymes found in cells (>11MDa). It catalyzes the production of acetyl-CoA from pyruvate and is comprised of three component enzymes: E1, E2, and E3. Previous work from our laboratory resulted in determination of the molecular architectures of sub-complexes comprising the 60-mer E2 from Bacillus stearothermophilus decorated with either 60 copies of E1 or E3. An annular gap of ~75–95 Å separates the catalytic domains of the E2 from an outer shell formed of E1’s or E3’s. To test the mechanisms involved in the maintenance of this annular gap and its significance for active site coupling, cryo-electron tomography was employed to determine the spatial relationship of E1 and E2 components within individual, non-averaged, E1E2 sub-complexes. In addition to verifying the presence of the annular space in three dimensions, stochastic variation in the exact position of each individual E1 density relative to the E2 core was measured. Biophysical studies of a peptide, corresponding to the inner linker region, suggests this region is flexible, yet highly extended, and maintains a long persistence length (~70–89 Å). Together these results demonstrate that the inner linker region separating the bound E1 and E3 molecules from the E2 core is an extended peptide chain that maintains a long persistence length, sixty of which generate a scaffold for the organization of E1 and E3 molecules in a modular radial shell. This is likely to be critically important to the coupling of E1, E2, and E3 active sites, mediated by movements across the gap by an additional domain at the N-terminal end of the E2 chain.

Molecular architecture of the pyruvate dehydrogenase complex: bridging the gap

The PDC (pyruvate dehydrogenase complex) is a high-molecular-mass (4-11 MDa) complex of critical importance for glucose homoeostasis in mammals. Its multi-enzyme structure allows for substrate channelling and active-site coupling: sequential catalytic reactions proceed through the rapid transfer of intermediates between individual components and without diffusion into the bulk medium due to its 'swinging arm' that is able to visit all PDC active sites. Optimal positioning of individual components within this multi-subunit complex further affects the efficiency of the overall reaction and stability of its intermediates. Mammalian PDC comprises a 60-meric pentagonal dodecahedral dihydrolipoamide (E2) core attached to which are 30 pyruvate decarboxylase (E1) heterotetramers and six dihydrolipoamide (E3) homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein associated with the E2 core. Association of the peripheral E1 and E3 enzymes with the PDC core has been studied intensively in recent years and has yielded some interesting and substantial differences when compared with prokaryotic PDCs.

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.

Active site coupling in multienzyme complexes

Active site coupling in multienzyme complexes

The Molecular Origins of Specificity in the Assembly of a Multienzyme Complex

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.

Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine.

Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.

Corrigendum

Corrigendum1 November 2002free access Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor Dean J. Derbyshire Dean J. Derbyshire Search for more papers by this author Balaku P. Basu Balaku P. Basu Search for more papers by this author Louise C. Serpell Louise C. Serpell Search for more papers by this author Woo S. Joo Woo S. Joo Search for more papers by this author Takayasu Date Takayasu Date Search for more papers by this author Kuniyoshi Iwabuchi Kuniyoshi Iwabuchi Search for more papers by this author Aidan J. Doherty Aidan J. Doherty Search for more papers by this author Dean J. Derbyshire Dean J. Derbyshire Search for more papers by this author Balaku P. Basu Balaku P. Basu Search for more papers by this author Louise C. Serpell Louise C. Serpell Search for more papers by this author Woo S. Joo Woo S. Joo Search for more papers by this author Takayasu Date Takayasu Date Search for more papers by this author Kuniyoshi Iwabuchi Kuniyoshi Iwabuchi Search for more papers by this author Aidan J. Doherty Aidan J. Doherty Search for more papers by this author Author Information Dean J. Derbyshire, Balaku P. Basu, Louise C. Serpell, Woo S. Joo, Takayasu Date, Kuniyoshi Iwabuchi and Aidan J. Doherty The EMBO Journal (2002)21:5953-5953https://doi.org/10.1093/emboj/cdf615 This article corrects the following: Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor15 July 2002 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The EMBO Journal, 21, 3863–3872, 2002 Next ArticlePrevious Article Read MoreAbout the coverClose modalView large imageVolume 21,Issue 21,November 1, 2002Cover. Sectioned schematic view of an icosahedral pyruvate dehydrogenase complex based on electron cryo-microscopic analysis of an E1E2 sub-complex from B.stearothermophilus. Three of the 60 E2 molecules (colored red, green and yellow) are highlighted. The movement of the swinging E2 lipoyl domain in the annular region between the inner core (cyan) of E2 molecules and the outer shell of E1 molecules (purple) is proposed to be a critical feature underlying active site coupling in the complex. Jacqueline Milne and Sriram Subramaniam work at the National Cancer Institute, NIH in Bethesda, MD, USA ( http://hrem.nci.nih.gov). The artist is Donald Bliss at the Medical Arts and Photography Branch at NIH. For further details, see Milne et al., pp. 5587--5598. Volume 21Issue 211 November 2002In this issue RelatedDetailsLoading ...

The complex fate of alpha-ketoacids.

Plant cells are unique in that they contain four species of alpha-ketoacid dehydrogenase complex: plastidial pyruvate dehydrogenase, mitochondrial pyruvate dehydrogenase, alpha-ketoglutarate (2-oxoglutarate) dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. All complexes include multiple copies of three components: an alpha-ketoacid dehydrogenase/decarboxylase, a dihydrolipoyl acyltransferase, and a dihydrolipoyl dehydrogenase. The mitochondrial pyruvate dehydrogenase complex additionally includes intrinsic regulatory protein-kinase and -phosphatase enzymes. The acyltransferases form the intricate geometric core structures of the complexes. Substrate channeling plus active-site coupling combine to greatly enhance the catalytic efficiency of these complexes. These alpha-ketoacid dehydrogenase complexes occupy key positions in intermediary metabolism, and a basic understanding of their properties is critical to genetic and metabolic engineering. The current status of knowledge of the biochemical, regulatory, structural, genomic, and evolutionary aspects of these fascinating multienzyme complexes are reviewed.

Substrate channelling in 2-oxo acid dehydrogenase multienzyme complexes

Heteronuclear NMR spectroscopy and other experiments indicate that the true substrate of the E1 component of 2-oxo acid dehydrogenase complexes is not lipoic acid but the lipoyl domain of the E2 component. E1 can recognize the lipoyllysine residue as such, but reductive acylation ensues only if the domain to which the lipoyl group is attached is additionally recognized by virtue of a mosaic of contacts distributed chiefly over the half of the domain that contains the lipoyl-lysine residue. The lipoyl-lysine residue may not be freely swinging, as supposed hitherto, but may adopt a preferred orientation pointing towards a nearby loop on the surface of the lipoyl domain. This in turn may facilitate the insertion of the lipoyl group into the active site of E1, where reductive acylation is to occur. The results throw new light on the concept of substrate channelling and active-site coupling in these giant multifunctional catalytic machines.