NADH Binding Research Articles

Dynamics of NADH Cofactor Binding to B. Stearothermophilus Lactate Dehydrogenase

Laser induced temperature jump relaxation spectroscopy was used to study NADH nucleotide cofactor binding to lactate dehydrogenase from Bacillus Stearothermophilus thermophylic bacteria (bsLDH). The tryptophan fluorescence T-jump kinetics, that are determined almost exclusively by NADH binding/unbinding, show only one exponential relaxation component, while the NADH fluorescence T-jump kinetics, that report on NADH (un)binding and conformation changes around the active site, display three distinct relaxation components. The mid-range NADH relaxation rates are very close to the relaxation rates of the tryptophan kinetics, and their dependence on the total concentration of free ligand and enzyme shows a sharp initial increase and much slower rise at higher concentrations. The slow NADH relaxation rates are nearly independent of the total free ligand and enzyme concentration. Our kinetic data can be best described by a model in which NADH binding to the binding pocket of bsLDH is followed by a conformation change, presumably closing of the active site loop, and additional weak NADH binding to the binary complex bsLDH·NADH in the open-loop conformation only. The presumed open and closed loop conformations, that can be considered as competent and incompetent for further substrate binding, interconvert with relaxation time of about 1 ms.

Structural Basis of Free Reduced Flavin Generation by Flavin Reductase from Thermus thermophilus HB8

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.

Tyrosine Phosphorylation of Lactate Dehydrogenase A Is Important for NADH/NAD+ Redox Homeostasis in Cancer Cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.

The Reaction Mechanism of Cytochrome P450 NO Reductase: A Detailed Quantum Mechanics/Molecular Mechanics Study

A detailed QM/MM study on the reaction mechanism of Cytochrome P450 NO reductase is reported. Two reaction pathways connecting the two well-characterized intermediates as well as two putative intermediates that represent the unknown third intermediate are explored, with emphasis on the unusual direct reduction of the enzymatic active site by the cofactor NADH. Activation barriers and kinetic isotope effect are calculated and reveal that reduction of the NO-bound species occurs in form of a hydride ion transfer. Furthermore, the impact of different hydrogen bonds in the active site to binding and reactivity of NADH is explored. The calculated kinetic and thermodynamic properties for both modelled pathways are used for the kinetic simulation of the entire reaction course. It is thus shown that the unknown key intermediate is the singlet diradical Fe(III)-NHOH(⋅). It is also found that the mechanism of the N-N bond formation is spin-recoupling, which is only possible due to the diradical character of the key intermediate.

Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.

Genetically Encoded Fluorescent Sensors for Intracellular NADH Detection

We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile.

Mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. It has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (NTA-Mo). NTA-Mo is a member of a class of enzymes which consist of two components: A and B. While component A has monooxygenase activity and is responsible for the oxidation of the substrate, component B consumes cofactor to generate reduced flavin mononucleotide, which is required for component A activity. NTA-MoB from M. thermoresistibile, a rare but infectious close relative of M. tuberculosis which can thrive at elevated temperatures, has been expressed, purified and crystallized. The 1.6 Å resolution crystal structure of component B of NTA-Mo presented here is one of the first crystal structures determined from the organism M. thermoresistibile. The NTA-MoB crystal structure reveals a homodimer with the characteristic split-barrel motif typical of flavin reductases. Surprisingly, NTA-MoB from M. thermoresistibile contains a C-terminal tail that is highly conserved among mycobacterial orthologs and resides in the active site of the other protomer. Based on the structure, the C-terminal tail may modulate NTA-MoB activity in mycobacteria by blocking the binding of flavins and NADH.

Contribution of K99 and D319 to substrate binding and catalysis in the saccharopine dehydrogenase reaction

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to l-lysine and α-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the α-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V 2/ K m values for lysine, α-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/ K Lys increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V 2/ K NADH by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and α-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH.

The Evolution of Respiratory Chain Complex I from a Smaller Last Common Ancestor Consisting of 11 Protein Subunits

The NADH:quinone oxidoreductase (complex I) has evolved from a combination of smaller functional building blocks. Chloroplasts and cyanobacteria contain a complex I-like enzyme having only 11 subunits. This enzyme lacks the N-module which harbors the NADH binding site and the flavin and iron–sulfur cluster prosthetic groups. A complex I-homologous enzyme found in some archaea contains an F420 dehydrogenase subunit denoted as FpoF rather than the N-module. In the present study, all currently available whole genome sequences were used to survey the occurrence of the different types of complex I in the different kingdoms of life. Notably, the 11-subunit version of complex I was found to be widely distributed, both in the archaeal and in the eubacterial kingdoms, whereas the 14-subunit classical complex I was found only in certain eubacterial phyla. The FpoF-containing complex I was present in Euryarchaeota but not in Crenarchaeota, which contained the 11-subunit complex I. The 11-subunit enzymes showed a primary sequence variability as great or greater than the full-size 14-subunit complex I, but differed distinctly from the membrane-bound hydrogenases. We conclude that this type of compact 11-subunit complex I is ancestral to all present-day complex I enzymes. No designated partner protein, acting as an electron delivery device, could be found for the compact version of complex I. We propose that the primordial complex I, and many of the present-day 11-subunit versions of it, operate without a designated partner protein but are capable of interaction with several different electron donor or acceptor proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00239-011-9447-2) contains supplementary material, which is available to authorized users.

Abstract 2798: Tyrosine phosphorylation of LDH-A is important for cancer cell redox homeostasis and tumor growth

Abstract Cancer cells take up more glucose than normal tissue and favor aerobic glycolysis, generating lactate through a NADH-dependent enzyme, lactate dehydrogenase A (LDH-A). This is the last step of glycolysis that permits the regeneration of NAD+, which is needed as an electron acceptor to maintain cytosolic glucose catabolism. Therefore, most tumor cells are reliant on lactate production for their survival. LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production, and expression of LDH-A was previously implicated to be involved in tumour initiation and growth. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. Our phospho-proteomics studies revealed that oncogenic fibroblast growth factor (FGF) receptor type 1 (FGFR1) tyrosine kinase directly phosphorylates LDH-A. Structural and biochemical studies revealed that phosphorylation at Y10 and Y83 activates LDH-A by promoting the formation of active, tetrameric LDH-A and binding of LDH-A substrate NADH, respectively. Moreover, we found that LDH-A is commonly phosphorylated at Y10 in diverse human cancer cells by multiple oncogenic tyrosine kinases including BCR-ABL, FLT3 and JAK2, which represents an acute molecular mechanism underlying increased lactate production in cancer cells. Furthermore, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, show decreased cell proliferation and ATP levels under hypoxia, increased mitochondrial respiration to sustain glycolysis by providing NAD+, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation activates LDH-A to promote the Warburg effect and tumor growth by regulating NADH/NAD+ redox homeostasis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2798. doi:10.1158/1538-7445.AM2011-2798

Small-angle X-ray Scattering Study of a Rex Family Repressor: Conformational Response to NADH and NAD+ Binding in Solution

The transcriptional repressor Rex is a sensor of the intracellular NADH/NAD+ redox state through direct binding of NADH or NAD+. Homodimeric Rex protein from Thermus aquaticus (T-Rex) and Bacillus subtilis (B-Rex) exists in several different conformations. In both organisms, Rex in complex with NADH has the DNA binding domains packed together at the dimer interface, whereas in the apo form of B-Rex the linkers connecting these domains to the core are flexible. The crystal structures of the apo forms of B-Rex and a mutated variant of T-Rex are radically different. We describe the solution structures of B-Rex in complex with NAD+ or NADH and in its apo form, on the basis of small-angle X-ray scattering (SAXS) measurements. This study addresses to what extent the unusual orientation of the DNA recognition domains of the crystal structure of apo B-Rex is due to stabilization by crystal packing. Low-resolution ab initio solution structures were obtained for apo B-Rex, B-Rex:NADH and B-Rex:NAD+. Models giving a more detailed picture of these three solution structures were obtained also by rigid body fitting of the crystallographic domains. The SAXS data confirm the elongated and flexible nature of apo-B-Rex and the existence of two distinct and more rigid conformations for the complexes with NADH and NAD+. The models emerging from this study indicate a reaction mechanism for B-Rex in which the recognition domains are rotated upon binding to NADH.

Molecular basis of two novel mutations found in type I methemoglobinemia

Congenital methemoglobinemia due to NADH–cytochrome b5 reductase 3 (CYB5R3) deficiency is an autosomal recessive disorder that occurs sporadically worldwide, although endemic clusters of this disorder have been identified in certain ethnic groups. It is present as two distinct phenotypes, type I and type II. Type I methemoglobinemia is characterized by CYB5R3 enzyme deficiency restricted to erythrocytes and is associated with benign cyanosis. The less frequent type II methemoglobinemia is associated with generalized CYB5R3 deficiency affecting all cells and is lethal in early infancy. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients from three distinct ethnic backgrounds, Asian Indian, Mexican and Greek. The CYB5R3 gene of three probands with type I methemoglobinemia and their relatives were sequenced revealing several putative causative mutations; in one subject multiple mutations were present. Two novel mutations, S54R and F157C, were identified and the previously described A179T, V253M mutations were also identified. All these point mutations mapped to the NADH binding domain and or the FAD binding domain. Each has the potential to sterically hinder cofactor binding causing instability of the CYB5R3 protein. Wild-type CYB5R3, as well as two of these novel mutations, S54R and F157C, was amplified, cloned, and purified recombinant peptide obtained. Kinetic and thermodynamic studies of these proteins show that the above mutations lead to decreased thermal stability.

Assessing the Metabolic State of Cultured Cells and the Intact Cochlea by Two-Photon Fluorescence Intensity and Lifetime Imaging

The ability to match energy production with demand is a fundamental determinant of the health of cells, tissues, and organs of the body. Metabolism and mitochondrial dysfunction have been broadly implicated in many disease states, including hearing disorders and noise-induced hearing loss. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) and flavoprotein (Fp) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, the fluorescence intensity has commonly been used as an indicator of the reduced NADH and/or oxidized Fp concentration in cells and tissue. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. This presents a new opportunity to track the cellular metabolic state as NADH binding changes with metabolism. To better understand what is revealed by variations in the fluorescence intensity and lifetime distributions, we have used mitochondrial uncouplers and inhibitors in conjunction with manipulation of substrate and oxygen concentrations to systematically adjust the metabolic state. We will compare the similarities and differences of the intensity- and FLIM-based assessment of metabolic rate in model systems of increasing complexity, ranging from single cells to intact tissue, highlighting the advantages and limitations of each technique. Supported by NIH DC 02053, NSF-EPSCoR EPS-0701892 (CFD 47.076), NIH P20 RR16469 from the National Center for Research Resources (NCRR), and NIH R15 GM085776.

Docking Studies of Chloroquine Binding to Plasmodium Falciparum Glyceraldehyde-3-Phosphate Dehydrogenase

Malaria is a major infectious disease affecting millions of people worldwide annually. Plasmodium falciparum is the most virulent of the malaria parasites and its resistance to currently available drugs continues to grow and presents an impediment to attempts to completely contain this disease. Considering that the malaria parasite relies mainly on glycolysis for its energy needs, enzymes of the glycolytic pathway are emerging as potential targets for new antimalarial drugs.Chloroquine is an antimalarial drug that has been shown to bind to the NADH cofactor binding site of Plasmodium falciparum lactate dehydrogenase (LDH). This suggests that chloroquine and NADH are competitive inhibitors for this particular enzyme. In this study, molecular docking was used to determine and explore potential binding of chloroquine to Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (pfGAPDH), another glycolytic enzyme that shares a common cofactor with LDH. Automated docking using Autodock 4.2 was employed to determine binding sites and binding energies of chloroquine docked to monomeric and tetrameric forms of pfGAPDH with and without the cofactor present.Docking solutions for the monomer revealed two binding sites for chloroquine, one of which was the cofactor binding pocket. In the absence of NADH, 61 % of conformations with sufficiently low binding energy were located in the NADH binding pocket (average binding energy of −5.943 ± 0.085 kcal/mol) with 31% in the secondary binding site (average binding energy of −4.937 ± 0.025 kcal/mol). With the cofactor present, the secondary binding pocket accounted for 100% of stable complexes with an average binding energy of −4.803± 0.015 kcal/mol. With the tetramer, the most stable complexes were found in a new hydrophobic pocket at the interface between the subunits with 56% and 80% favorability in the absence and presence of cofactor, respectively.

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg 2+ ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2–NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes ( τ) of free NADH ( τ = 0.4 ns) and bound NADH ( τ = 6.0 ns). We used this technique to investigate the effects of Mg 2+ on the ALDH2–NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K D for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg 2+ ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme–NADH complex ranged from 0.4 s −1 (6000 μM Mg 2+) to 8.3 s −1 (0 μM Mg 2+) as determined by addition of excess NAD + to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 μM −1 s −1 over the entire Mg 2+ ion concentration range and demonstrate that Mg 2+ ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg 2+ concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2–NADH complexes corresponding to free NADH, enzyme–bound NADH, and, potentially, an abortive NADH–enzyme–propanal complex ( τ = 11.2 ns).

Characterization of specific binding by mass spectrometry: Associations of E. coli citrate synthase with NADH and 2-azidoATP

Type II citrate synthases (CSs), found in gram-negative bacteria such as Escherichia coli, are subject to strong and specific allosteric inhibition by NADH. Crystallographic studies have shown that the NADH binding sites are located at macromolecular contacts, so that tight binding requires the intact hexameric structure. Although other adenylates, such as 5′-AMP and ATP, are known to influence CS function by inhibiting NADH binding, the exact mechanism of inhibition is currently unknown. In the present study, we have used mass spectrometry to study the adenylate-binding properties of CS by ESI and MALDI-based techniques. More specifically, to investigate where adenylates bind, we have used the photoaffinity label, 2-azidoATP, to label E. coli CS under non-denaturing conditions for MS analysis of tryptic peptides. Different MS techniques are used to show that tryptic peptide T17 (AA168–177) of the CS sequence known to contribute to NADH binding, disappears after photolabeling, while a new photoaffinity-labeled peptide, with a mass 520 Da than that of T17, appears instead. Another new peptide, corresponding to photolabeled peptide T16–18 (a combined peptide arising from incomplete digestion of the same region) has also been detected. By CE-ESI-TOF-MS, ESI-Q 3, MALDI-TOF and analysis of complex stoichiometry we conclude that ATP/ATP-analogs, and presumably the whole series of adenylates potentiate CS and the tricarboxylic acid cycle through the identified site of NADH regulation.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4Å resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.

Comparative Kinetics of Cofactor Association and Dissociation for the Human and Trypanosomal S-Adenosylhomocysteine Hydrolases. 3. Role of Lysyl and Tyrosyl Residues of the C-Terminal Extension

On the basis of the available X-ray structures of S-adenosylhomocysteine hydrolases (SAHHs), free energy simulations employing the MM-GBSA approach were applied to predict residues important to the differential cofactor binding properties of human and trypanosomal SAHHs (Hs-SAHH and Tc-SAHH), within 5 Å of the cofactor NAD(+)/NADH binding site. Among the 38 residues in this region, only four are different between the two enzymes. Surprisingly, the four nonidentical residues make no major contribution to differential cofactor binding between Hs-SAHH and Tc-SAHH. On the other hand, four pairs of identical residues are shown by free energy simulations to differentiate cofactor binding between Hs-SAHH and Tc-SAHH. Experimental mutagenesis was performed to test these predictions for a lysine residue and a tyrosine residue of the C-terminal extension that penetrates a partner subunit to form part of the cofactor binding site. The K431A mutant of Tc-SAHH (TcK431A) loses its cofactor binding affinity but retains the wild type's tetrameric structure, while the corresponding mutant of Hs-SAHH (HsK426A) loses both cofactor affinity and tetrameric structure [Ault-Riche, D. B., et al. (1994) J. Biol. Chem. 269, 31472-31478]. The tyrosine mutants HsY430A and TcY435A alter the NAD(+) association and dissociation kinetics, with HsY430A increasing the cofactor equilibrium dissociation constant from approximately 10 nM (Hs-SAHH) to ∼800 nM and TcY435A increasing the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to ∼1 mM. Both changes result from larger increases in the off rate combined with smaller decreases in the on rate. These investigations demonstrate that computational free energy decomposition may be used to guide experimental studies by suggesting sensitive sites for mutagenesis. Our finding that identical residues in two orthologous proteins may give significantly different binding free energy contributions strongly suggests that comparative studies of homologous proteins should investigate not only different residues but also identical residues in these proteins.

Insights into the Mechanism of Ligand Binding to Octopine Dehydrogenase from Pecten maximus by NMR and Crystallography

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD+, and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail.

NADH binding to complex I: Implications for the mechanism

NADH binding to complex I: Implications for the mechanism