Interaction of Fragment A from Diphtheria Toxin with Nicotinamide Adenine Dinucleotide

Kandel, Judith; Collier, R. J.; Chung, Dominic W.

doi:10.1016/s0021-9258(19)42800-7

Cited by 142 publications

(26 citation statements)

References 22 publications

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“…Additionally, dissociation constants for NAD binding to the mutant and wild-type toxins at pH 8.0 and 25 °C were measured independently of enzymatic activity by monitoring the decrease in intrinsic protein fluorescence at 335 nm (excitation at 285 nm) as a function of NAD concentration (Figure 3) and subjecting the data to Scatchard analysis after correction for the inner filter effect. The K6 value obtained for NAD binding to the wild-type DTA, 9.3 /*M, was in agreement with values published earlier (Kandel et al, 1974;Chung & Collier, 1977;Collier et al, 1974). The Kd values for NAD binding to the mutants E148D, E148Q, and E148S of 8.7, 12.7, and 12.6 uM, respectively, were close to that of the wild-type DTA and are consistent with the Km values obtained for the ADP-ribosylation and NAD hydrolysis reactions.…”

Section: Resultssupporting

confidence: 91%

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

et al. 1990

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Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 91%

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

et al. 1990

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“…Adenine binds to DTA at its active site, stabilizing its native conformation (Kandel et al, 1974), so that adenine in the cis solution should anchor the C-terminal end of LF N -DTA to the cis side of the membrane and thereby inhibit unblocking of LF N -DTA at large positive voltages. This is precisely what we saw (Fig.…”

Section: Diphtheria Toxin a Chain (Dta) Attached To The C-terminus Of Lf N (Lf N -Dta)mentioning

confidence: 99%

Protein Translocation through Anthrax Toxin Channels Formed in Planar Lipid Bilayers

Zhang

Udho

et al. 2004

Biophysical Journal

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105

146

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The 63-kDa fragment of the protective antigen (PA) component of anthrax toxin forms a heptameric channel, (PA63)7, in acidic endosomal membranes that leads to the translocation of edema factor (EF) and lethal factor (LF) to the cytosol. It also forms a channel in planar phospholipid bilayer membranes. What role does this channel play in the translocation of EF and LF? We report that after the 263-residue N-terminal piece of LF (LFN) binds to its receptor on the (PA63)7 channel and its N-terminal end enters the channel at small positive voltages to block it, LFN is translocated through the channel to the opposite side at large positive voltages, thereby unblocking it. Thus, all of the translocation machinery is contained in the (PA63)7 channel, and translocation does not require any cellular proteins. The kinetics of this translocation are S-shaped, voltage-dependent, and occur on a timescale of seconds. We suggest that the translocation process might be explained simply by electrophoresis of unfolded LFN through the channel, but the refolding of the N-terminal half of LFN as it emerges from the channel may also provide energy for moving the rest of the molecule through the channel.

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“…Although the regulation of dinitrogenase reductase by ADP-ribosylation is the best characterized reversible mono-ADP-ribosylation system, numerous other ADP-ribosylations are found in nature. The requirement for NAD for action led to the discovery that diphtheria toxin catalyzes the NAD-dependent ADP-ribosylation of the elongation factor involved in eukaryotic protein synthesis (Honjo et al, 1968;Kandel et al, 1974). Other bacterial toxins, including cholera toxin and pertussis toxin, catalyze the ADP-ribosylations of the subunits of eukaryotic G proteins, leading to cell death (Moss and Vaughan, 1977;West et al, 1985).…”

Section: Other Adp-ribosylationsmentioning

confidence: 99%

Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

Nordlund

Ludden

Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria

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The photosynthetic bacteria are remarkable for their range of metabolic capabilities and, as a result, the ability to survive under a wide range of environmental conditions, including nitrogen limitation. The photosynthetic bacteria are members of the alpha subdivision of the proteobacteria (Imhoff et al., 1984;Woese et al., 1984) and, with a single known exception, the wild-type strains of all purple sulfur bacteria and all purple non-sulfur bacteria are capable of N 2 fixation (Madigan et al., 1984). Most of the work that will be described in this chapter has been performed on a few species of purple, non-sulfur bacteria, including Rhodospirillum rubrum and Rhodobacter capsulatus. This chapter will focus on the biochemistry and physiology of N 2 fixation by photosynthetic bacteria and the regulation of nitrogenase activity by reversible ADP-ribosylation of the dinitrogenase reductase (Fe protein). ADP-ribosylation has also been found in Azospirillum and other genera and, where relevant, that work will be included. Other chapters in this series will cover the genes of the nif, anf and rnf regulons of the photosynthetic bacteria and the regulation of the expression of those genes. Although this chapter will discuss aspects of the nitrogenase enzyme, the mechanistic and structural details of nitrogenase will be covered in more detail in Volume 1 of this series.

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Interaction of Fragment A from Diphtheria Toxin with Nicotinamide Adenine Dinucleotide

Cited by 142 publications

References 22 publications

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

Protein Translocation through Anthrax Toxin Channels Formed in Planar Lipid Bilayers

Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

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