[52] Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: Techniques and applications

Potter, Robert; Haley, Boyd E.

doi:10.1016/s0076-6879(83)91054-6

Cited by 138 publications

(72 citation statements)

References 26 publications

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“…An analysis of the tryptic peptides showed a new labeled peptide (in addition to the one labeled at 20 pM 8-azido-AMP) corresponding to residues Val279 -Lys296 (Val-ValThr-Glu-Gly-Ala-Gly-Ala-Leu-Ala-Cys-Ala-Ala-Leu-LeuSer-Gly-Lys) on the protein primary structure. These findings are contrary to the general expectation [24] of random nonspecific incorporation of labeled azido-purine analogs into proteins at high ligand concentrations. In view of this, the significance of selective covalent attachment of 8-azido-AMP at the 'second-site' peptide remains to be uncovered.…”

Section: Identification Of the 8-azido-amp Binding Sitecontrasting

confidence: 99%

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Brand

Heinickel

Kratzin

et al. 1988

European Journal of Biochemistry

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The photoreactive AMP analog, g-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the K , for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 pM and 1.5 pM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Since the initial report [ 13 that the biodegradative threonine dehydratase of Escherichia coli, which catalyzes the pyridoxal phosphate-dependent dehydration of threonine and serine to ammonia and corresponding 2-0x0 acids, is stimulated by AMP, considerable evidence has been gathered on the effect of this nucleotide on the conformational states, catalytic mechanism, and regulation of enzyme activity by various cellular metabolites. For example, the enzyme can bind 4 mol AMP/mol protein [2], and the AMP-free enzyme exists as a protomer of M , 35000, whereas the AMP-bound form is a tetramer of M , 140000 which exhibits a 25-fold decrease in the K , for threonine [2-41. The stimulatory effect of AMP has been correlated with obligatory oligomerization of the protomeric species [4]. Furthermore, allosteric inhibition of enzyme activity [5 -71 and catabolite inactivation by covalent protein modification of the dehydratase by intermediary metabolites [7 -91 are known to be precisely regulated by subtle variations in the concentration of AMP. Nevertheless, the binding site for AMP on the enzyme molecule remains unidentified. Recently, the complete amino acid sequence of the E. coli threonine dehydratase has been determined in this laboratory from the nucleotide sequence of the cloned gene [lo, 111, and the active-site pyridoxal-phosphate-bound lysine residue involved in the dehydration reaction has been localized on the protein primary structure [ I l , 121. This report is concerned with photolabeling of the binding site of 8-azido-AMP, a photoaffinity analog of AMP. The experiments revealed that 8-azido-AMP can mimic the allosteric modifier AMP and binds to a unique site on the dehydratase molecule which most likely represents the binding region of AMP.

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Section: Identification Of the 8-azido-amp Binding Sitecontrasting

confidence: 99%

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Brand

Heinickel

Kratzin

et al. 1988

European Journal of Biochemistry

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“…Vol. 103, 1993 N3ATP and [y-32P]ATPyBP were synthesized as described previously (Potter and Haley, 1983;Rajagopalan et al, 1993).…”

Section: Salvuccimentioning

confidence: 99%

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Salvucci¹

1993

Plant Physiol.

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Chemical modification of ribulose-1,5-bisphosphate carboxylaseloxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. lncubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-' s-l compared to 21 M-' s-' for sulfo-NHS-acetate. lnactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mo1 of AMCA per mo1 of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acidlysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of KZ4' accounted for 1 mo1 of AMCA incorporated per mo1 of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS-and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the y-phosphate showed that KZd7 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of KZ4' is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.

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“…We found it essential to keep the mercaptoethanol concentration at 370/~M otherwise the azido grouping in the molecule is converted resulting in a photo-insensitive derivative of [7-32p]N3ATP, which requires separation by chromatography [10].…”

Section: Discussionmentioning

confidence: 99%

“…Enzymatic methods for the synthesis of [7-32p]NaATP have been described [9,10]. They are based on exchange reactions [9] or require subsequent chromatographic purification of the product [10] and thus do not lend themselves easily to routinely producing carrier-free products.…”

Section: Introductionmentioning

confidence: 99%

Carrier‐free 8‐azidoadenosine 5′‐[γ‐³²P]triphosphate

Sabbatini

Holt

1987

FEBS Letters

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We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[y-32p]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[?,-32p]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.

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[52] Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: Techniques and applications

Cited by 138 publications

References 26 publications

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Carrier‐free 8‐azidoadenosine 5′‐[γ‐³²P]triphosphate

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[52] Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: Techniques and applications

Cited by 138 publications

References 26 publications

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Carrier‐free 8‐azidoadenosine 5′‐[γ‐32P]triphosphate

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Carrier‐free 8‐azidoadenosine 5′‐[γ‐³²P]triphosphate