Non-phosphorylated Substrates Research Articles

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other β-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular β-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other β-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other β-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis

Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.

Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-D-ribose 5-phosphate aldolase.

2-Deoxy-D-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERA(K12) (S238D/F200I/ΔY259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards D-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-D-ribose yield from 0.41 mol/mol D-glyceraldehyde to 0.81 mol/mol D-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-D-robose (DR), with a yield of 0.71 mol/mol D-glyceraldehyde and average productivity of 0.13 mol/L·h (17.94 g/L·h). These results demonstrate the potential for large-scale production of 2-deoxy-D-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-D-altrose from D-erythrose, as well as 2-deoxy-L-xylose and 2-deoxy-L-ribose from L-glyceraldehyde.

A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0-7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni(2+), Ba(2+) and Fe(2+). The apparent K (m) and V (max) values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min(-1) mg(-1), for 2-deoxyribose were 0.033 mM and 2.59 μmol min(-1) mg(-1), respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.

Identification and Validation of Inhibitor-Responsive Kinase Substrates Using a New Paradigm To Measure Kinase-Specific Protein Phosphorylation Index

Regulation of all cellular processes requires dynamic regulation of protein phosphorylation. We have developed an unbiased system to globally quantify the phosphorylation index for substrates of a specific kinase by independently quantifying phosphorylated and total substrate molecules in a reverse in-gel kinase assay. Non-phosphorylated substrate molecules are first quantified in the presence and absence of a specific stimulus. Total substrate molecules are then measured after complete chemical dephosphorylation, and a ratio of phosphorylated to total substrate is derived. To demonstrate the utility of this approach, we profiled and quantified changes in phosphorylation index for Protein Kinase CK2 substrates that respond to a small-molecule inhibitor. A broad range of inhibitor-induced changes in phosphorylation was observed in cultured cells. Differences among substrates in the kinetics of phosphorylation change were also revealed. Comparison of CK2 inhibitor-induced changes in phosphorylation in cultured cells and in mouse peripheral blood lymphocytes in vivo revealed distinct kinetic and depth-of-response profiles. This technology provides a new approach to facilitate functional analyses of kinase-specific phosphorylation events. This strategy can be used to dissect the role of phosphorylation in cellular events, to facilitate kinase inhibitor target validation studies, and to inform in vivo analyses of kinase inhibitor drug efficacy.

Structurally Informed Site-Directed Mutagenesis of a Stereochemically Promiscuous Aldolase To Afford Stereochemically Complementary Biocatalysts

2-Keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus is a highly thermostable type I aldolase that can catalyze carbon-carbon bond formation using nonphosphorylated substrates. However, it exhibits poor diastereocontrol in many of its aldol reactions, including the reaction of its natural substrates, pyruvate and D-glyceraldehyde, which afford a 55:45 mixture of D-2-keto-3-deoxygluconate (D-KDGlu) and D-2-keto-3-deoxy-galactonate (D-KDGal). We have employed detailed X-ray crystallographic structural information of this aldolase bound to these diastereoisomeric aldol products to selectively target specific amino acids for mutation for the rapid creation of stereochemically complementary mutants that catalyze either (Re)- or (Si)-facial selective aldol reactions to afford either D-KDGlu or D-KDGal with good levels of diastereocontrol.

Redesigning the Active Site of Transaldolase TalB from Escherichia coli: New Variants with Improved Affinity towards Nonphosphorylated Substrates

Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X-ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site-directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D-fructose from DHA and d,l-GA by using an adjusted colour assay. The best results with respect to the synthesis of D-fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l-GA (K(M)=24 mM). We demonstrated that this double mutant can use D-GA, glycolaldehyde and the L-isomer, L-GA, as acceptor substrates. This resulted in preparative synthesis of D-fructose, D-xylulose and L-sorbose when DHA was used as donor. Hence, we engineered a DHA-dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.

Directed evolution of transketolase activity on non-phosphorylated substrates

We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form l-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.

Per(6-guanidino-6-deoxy)cyclodextrins: synthesis, characterisation and binding behaviour toward selected small molecules and DNA

Per(6-guanidino-6-deoxy)-cyclodextrins , and are novel derivatives, resulting from homogeneous introduction of the guanidino group at the primary side of alpha-, beta- and gamma-cyclodextrins. The products were obtained from the corresponding amino derivatives, as direct guanidinylation of the known bromo-cyclodextrins provided mixtures. The new compounds were fully characterized by NMR spectroscopy and other analytical methods, and their interaction with guest molecules was studied. Strong complexation with 4-nitrophenyl phosphate () disodium salt was observed (K(binding) approximately 5 x 10(4) M(-1)), whereas the non-phosphorylated substrate nitrobenzene () formed a very weak complex. 2D ROESY spectra revealed cavity inclusion in both cases, however the orientation of was opposite to that of , such that the phosphate group is oriented toward the primary side facing the guanidine groups. The strong affinity of towards the phosphorylated guest suggested that interaction with DNA was possible. The new compounds were found to completely inhibit the migration of ultra pure calf thymus DNA during agarose gel electrophoresis, whereas no effects were observed with guanidine alone or with the plain cyclodextrins. Further, the condensation of DNA into nanoparticles in the presence of was demonstrated by atomic force microscopy, confirming strong electrostatic interaction between the biopolymer and the multicationic products . The strong guanidine-phosphate interactions between and DNA were therefore attributed to the clustering of the guanidine groups in the primary area of the cyclodextrin. Cavity effects could not be assessed.

Crystal Structure of d-Psicose 3-epimerase from Agrobacterium tumefaciens and its Complex with True Substrate d-Fructose: A Pivotal Role of Metal in Catalysis, an Active Site for the Non-phosphorylated Substrate, and its Conformational Changes

d-Psicose, a rare sugar produced by the enzymatic reaction of d-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of d-fructose and d-psicose by epimerizing the C-3 position, with marked efficiency for d-psicose. The crystal structures of DPEase and its complex with the true substrate d-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of d-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the β4–α4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound d-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.

The linoleic acid derivative DCP-LA selectively activates PKC-ɛ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

Isolation of phosphopeptides using solid phase enrichment

Protein phosphorylations are post-translational protein modifications that are crucial for intercellular signal transduction and the regulation of many cellular events. We describe herein a novel technology utilizing α-diazo functionalized solid phase resins to isolate phosphorylated peptides from non-phosphorylated substrates.

Structure-Based mutagenesis approaches toward expanding the substrate specificity of d-2-Deoxyribose-5-phosphate aldolase

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and d-glyceraldehyde-3-phosphate to generate d-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 Å crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, d-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor™. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT ( Δace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.

An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates

Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 °C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C6 to C3 aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S. solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme. The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S. solfataricuscells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 °C of 2.5 h, and is equally active with both D- and L-glyceraldehyde. It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH4.

An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates

Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C(6) to C(3) aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S. solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme. The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S. solfataricus cells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equally active with both d- and l-glyceraldehyde. It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH(4).

Purification and characterization of the pyridoxol-5′-phosphate: oxygen oxidoreductase (deaminating) from Escherichia coli

The E. coli gene pdxH encoding pyridoxol-5′-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al. (Kohara, Y., Akiyama, K. and Isono K. (1987) Cell 50, 495–508) and assigned to a stretch between 36.0 and 36.1 min of the E. coli chromosome. The gene was overexpressed as a MBP/PdxH fusion protein. The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease ‘factor Xa’ resulting in homogeneous PdxH protein after another column chromatography. Both the MBP/PdxH fusion protein and the PdxH protein were characterized. Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate. K m values of both proteins were similar ranging from 350 to 400 μM for the two substrates. The enzymes did not accept non-phosphorylated substrates. Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited ( K i 8 μM) by pyridoxal phosphate as a mixed type inhibitor. As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer. No indication was found that the PdxH protein forms a dimer. The monomer was shown to be catalytically active.

Utilization of cellobiose and other β-D-glucosides in Agrobacterium tumefaciens

Agrobacterium tumefaciens strain C58 was able to utilize carbon from cellobiose and some other β-D-glucosides as efficiently as from glucose. β-D-glucoside utilization was partially inducible and the induction was subject to catabolite repression by glucose, independently of the presence of cyclic AMP in the medium. It was also independent of Ti plasmid-encoded functions. β-D-glucosides were hydrolysed by a single, cytoplasmic and constitutively expressed β-glucosidase, which was active on non-phosphorylated substrates and insensitive to glucose inhibition.

Auramine O as a conformational probe to study glyceraldehyde 3-phosphate dehydrogenase.

The interaction of yeast glyceraldehyde 3-phosphate dehydrogenase with a cationic fluorescent dye, auramine O, was studied by equilibrium dialysis, circular dichroism and fluorescence spectroscopy. Two equal and independent dye binding sites per tetrameric enzyme were detected with a dissociation constant of (4.5 ± 1) × 10−5 M. The maximum fluorescence of bound auramine O is at 515 nm, the excitation spectrum is characterized by the maxima at 380 nm and in the region of 460–470 nm. The complex of auramine O with the enzyme exhibits circular dichroism with a maximum at about 455 nm. The binding of NADH has no effect on the enzyme-dye interaction, whereas the oxidized coenzyme and nicotinamide mononucleotide displace auramine O from the protein. Nicotinamide mononucleotide was shown to induce conformational changes upon binding to the enzyme and to be capable of serving as a coenzyme in the dehydrogenase reaction. AMP, ADP and ADP-ribose form ternary complexes with the enzyme-dye binary complex. This results in a marked increase in fluorescence of the bound dye and was used to characterize the interaction between the enzyme and the adenine-containing NAD+ fragments. In the presence of glyceraldehyde 3-phosphate, the fluorescence of the bound auramine O is quenched, the dye-protein association constant remaining unchanged. Glyceraldehyde, a non-phosphorylated substrate analog, and glycerol 3-phosphate, a competitive inhibitor, are without any effect. Modification of the active-site cysteine-149 with iodoacetate does not affect the dye binding or fluorescence, whereas modification with iodoacetamide diminishes the fluorescence of the enzyme-bound dye without altering the dissociation constant. The effect of adenine-containing NAD+ fragments on the fluorescence of the enzyme-bound dye does not change upon modification of cysteine-149. Auramine O, added together with ADP-ribose, protects the apoenzyme against the inactivation due to modification of essential arginine residues. The effect is similar to that of NAD+ and is absent with auramine O alone or ADP-ribose alone.