Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue.

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety of NAD+ onto eucaryotic elongation factor 2 (EF-2). To study the ETA site of interaction with EF-2, an immobilized EF-2 binding assay was developed. This assay demonstrates that ETA, in the presence of NAD+, binds to immobilized EF-2. Additionally, diphtheria toxin was also found to bind to the immobilized EF-2 in the presence of NAD+. Comparative analysis was performed with a mutated form of ETA (CRM 66) in which a histidine residue at position 426 has been replaced with a tyrosine residue. This immunologically cross-reactive, ADP-ribosyl transferase-deficient toxin does not bind to immobilized EF-2, thus explaining its lack of ADPRT activity. ETA bound to immobilized EF-2 cannot bind the monoclonal antibody TC-1 which specifically recognizes the ETA epitope containing His426. Immunoprecipitation of native ETA by mAb TC-1 is only achieved by incubating ETA in the presence of NAD+. Diethyl pyrocarbonate modification of the His426 residue blocks ETA binding to EF-2 and prevents the binding of the TC-1 antibody. Analogs of NAD+ containing a reduced nicotinamide ring or modified adenine moieties cannot substitute for NAD+ in the immobilized binding assay. Collectively, these data support our proposal that the site of ETA interaction with EF-2 includes His426 and that a molecule of NAD+ is required for stable interaction.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Characterization of oxidized nicotinamide adenine dinucleotide (NAD(+)) analogues using a high-pressure-liquid-chromatography-based NAD(+)-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD(+))-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD(+)) and products (adenosine 5'-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD(+) analogue, 2'-F-ribo-NAD(+), was a competing substrate or a competitive inhibitor for this toxin. This NAD(+) analogue was hydrolyzed at a rate of 0.2% that of NAD(+) yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD(+) analogue, epsilon-NAD(+), is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD(+) as determined using HPLC. The rate of hydrolysis of epsilon-NAD(+) calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD(+)-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD(+) and its analogues.     

Histidine 21 is at the NAD+ binding site of diphtheria toxin

Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.     

The inhibition of pig kidney alkaline phosphatase by oxidized or reduced nicotinamide–adenine dinucleotide and related compounds

1. The inhibition of alkaline phosphatase by NAD(+), NADH, adenosine and nicotinamide was studied. 2. All of these substances except NAD(+) act as uncompetitive inhibitors, i.e. double-reciprocal plots are parallel. NAD(+), however, is a ;mixed' inhibitor of alkaline phosphatase and is less potent than NADH. 3. Inhibition studies with pairs of the inhibitors suggest that, in spite of the difference in type of inhibition, NAD(+) and NADH bind to alkaline phosphatase at a common site. Adenosine and nicotinamide also seem to bind at the NAD site and the binding of adenosine is facilitated by nicotinamide, and vice versa. 4. The facilitation may indicate the occurrence of an induced fit for NAD(+) and NADH. Attempts to desensitize alkaline phosphatase to NAD(+) and NADH inhibition by partial denaturation were unsuccessful. 5. The results are discussed in terms of a two-site model in which separate, but interacting, regions exist on the enzyme to accommodate the adenosine and nicotinamide moieties of NAD, and a single-site model in which the adenosine part of the molecule is bound preferentially and this interacts with the nicotinamide fraction. 6. The activity of alkaline phosphatase can be changed fourfold by alteration of the NAD(+)/NADH ratio. This sensitivity to the redox state of the coenzyme could be a means of controlling phosphatase activity.     

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.     

Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.     

Photolabelling of cholera toxin by NAD+.

When cholera toxin is incubated under u.v. light with NAD+ labelled in either the adenine or the nicotinamide moiety, radioactivity becomes covalently bound to the protein. The reaction is specific for cholera toxin, and is inhibited by excess unlabelled NAD+ or NAD analogues. Only the active A 1 chain of the toxin is labelled. The u.v.-absorption spectrum of the product is very similar to that of NAD+, and shows the same reaction with cyanide. The nature of the product is therefore different from that found when diphtheria toxin is photolabelled [Carroll & Collier (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3307-3311] in that the yield is lower, but both moieties of the NAD molecule become bound.     

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.     

Site in Cell-free Protein Synthesis Sensitive to Diphtheria Toxin

The effects of diphtheria toxin on cell-free protein synthesis in a bacterial system, and preparations obtained from animals that were sensitive and resistant to toxin were examined. In the presence of nicotinamide adenine dinucleotide (NAD), toxin inhibited the incorporation of amino acids by endogenous and synthetic polynucleotides in both rat liver and guinea pig liver cell-free systems that were exposed to 6 Lf units per ml of toxin. A cell-free system derived from Streptococcus faecalis was resistant to high concentrations of toxin. Dialyzed toxin-antitoxin floccules that are formed in the presence of NAD and the 105,000 x g supernatant fluid from rat liver contain NAD. Such floccules are also active in protein synthesis in the absence of added transferase I or II. An operational model presents the view that the intoxication complex is formed at the ribosomal level and occurs in two steps. First, the toxin molecule binds to transferase II and alters its stereospecific relationship to transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase II caused by the binding of diphtheria toxin allows NAD to bridge between transferase I and II, which then results in an inactivated complex. The sensitivity of the cell-free system derived from the normally resistant rat implies that in some cells the cell membrane serves as a permeability barrier to the toxin molecule. The resistance of bacterial cell-free protein synthesizing systems to diphtheria toxin may reflect basic differences between transferase enzymes from bacterial and mammalian sources.     

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.     

Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin

When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin. Potassium depletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L-cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells to bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.     

Specificity of coenzyme analogues and fragments in promoting or impeding the refolding of clostridial glutamate dehydrogenase

NAD+ facilitates high-yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea. The specificity of this effect has been explored by using analogues and fragments of NAD+. The adenine portion, unlike the nicotinamide portion, is important for reactivation. Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6-NH2 substitution on the adenine ring, in 6-deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding. Also ADP-ribose, lacking nicotinamide, promotes reactivation whereas NMN-phosphoribose, lacking the adenine, does not. Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding. These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase. It is assumed that the refolding intermediate has a more or less fully formed NAD+-binding domain but a partially disordered substrate-binding domain and linking region. Binding of NAD+ or ADP-ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.     

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.Abbreviations NADase nicotinamide adenine dinucleotide glycohydrolase - NAD nicotinamide adenine dinucleotide - ADP-ribose adenosine diphosphoribose - cADPR cyclic adenosine diphosphoribose     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

Interaction of nad with rape alcohol dehydrogenase

Rape alcohol dehydrogenase is competitively inhibited with respect to NAD by nicotinamide, as well as by compounds containing adenine (adenine, adenosine, AMP, ADP, ATP). Adenine and adenosine are bound more firmly to the enzyme than nicotinamide. The two types of compound, as component parts of the NAD coenzyme, are bound to different sites on the enzyme. Adenine and adenosine compete for the adenine nucleotide bonding site, but they do not compete for the o-phenanthroline bonding site. Nicotinamide competes with o-phenanthroline for the binding site at which the metal is apparently present.     

NAD(P)H oscillation in relation to the rhythmic contraction in thePhysarum plasmodium

Summary ThePhysarum plasmodium shows rhythmic contractile activities with a period of a few min. Phases of the oscillation in the plasmodium migrating unindirectionally agreed sideways throughout at the frontal part. So, time course of an intracellular chemical component was determined by analyzing small pieces cut off successively from the frontal part of the large plasmodium. Intracellular NAD(P)H concentration oscillated with the same period as the rhythmic contraction but with a different phase advancing about 1/3 of the period. UV irradiation suppressed the rhythmic contraction without affecting the rhythmic variation of NAD(P)H. Thus, the NAD(P)H oscillator works independently of the rhythmic contractile system, but seems entraining with each other.Abbreviations UV ultraviolet - NADH nicotinamide adenine dinucleotide, reduced form - NADPH nicotinamide adenine dinucleotide phosphate, reduced form - ATP adenosine 5-triphosphate - cAMP cyclic adenosine 3, 5-monophosphate - FMNH₂ flavin mononucleotide, reduced form - TCA tricarboxylic acid - BSA bovine serum albumin - DTT dithiothreitol     

An isothermal titration calorimetry study of the binding of substrates and ligands to the tartrate dehydrogenase from Pseudomonas putida reveals half-of-the-sites reactivity

An isothermal titration calorimetric study of the binding of substrates and inhibitors to different complexes of tartrate dehydrogenase (TDH) from Pseudomonas putida was carried out. TDH catalyzes the nicotinamide adenine dinucleotide (NAD)-dependent oxidative decarboxylation of d-malate and has an absolute requirement for both a divalent and monovalent metal ion for activity. The ligands Mn(2+), meso-tartrate, oxalate, and reduced nicotinamide adenine dinucleotide (NADH) bound to all TDH complexes with a stoichiometry of 1 per enzyme dimer. The exception is NAD, which binds to E/K(+), E/K(+)/Mn(2+), and E/K(+)/Mg(2+) complexes with a stoichiometry of two per enzyme dimer. The binding studies suggest a half-of-the-sites mechanism for TDH. No significant heat changes were observed for d-malate in the presence of the E/K(+)/Mn(2+) complex, suggesting that it did not bind. In contrast, meso-tartrate does bind to E/K(+)/Mn(2+) but gives no significant heat change in the presence of E/Mn(2+), suggesting that K(+) is required for meso-tartrate binding. meso-Tartrate also binds with a large DeltaC(p) value and likely binds via a different binding mode than d-malate, which binds only in the presence of NAD. In contrast to all of the other ligands tested, the binding of Mn(2+) is entropically driven, likely the result of the entropically favored disruption of ordered water molecules coordinated to Mn(2+) in solution that are lost upon binding to the enzyme. Oxalate, a competitive inhibitor of malate, binds with the greatest affinity to E/K(+)/Mn(2+)/NADH, and its binding is associated with the uptake of a proton. Overall, with d-malate as the substrate, data are consistent with a random addition of K(+), Mn(2+), and NAD followed by the ordered addition of d-malate; there is significant synergism in the binding of NAD and K(+). Although the binding of meso-tartrate also requires enzyme-bound K(+) and Mn(2+), the binding of meso-tartrate and NAD is random.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

The mechanism of ADP-ribosylation of elongation factor 2 catalyzed by fragment A from diphtheria toxin.

Measurements of the initial rate of ADP-ribosylation of elongation factor 2 (EF-2) catalyzed by Fragment A from diphtheria toxin support a sequential mechanism and suggest that the reaction proceeds through a central ternary complex involving Fragment A and the substrates, EF-2 and NAD. The Michaelis constants for EF-2 and NAD are 0.15 and 1.4 muM, respectively. As determined by equilibrium gel permeation, EF-2 does not bind Fragment A significantly, alone or in the presence of adenine, ADPribose, nicotinamide or NADH. Based on these and earlier results, we propose an ordered sequential mechanism for the reaction; the sequence of binding of substrates is NAD, followed by EF-2.