A photolabile reagent, p-azidobenzoyl-CoA, has been synthesized and tested as a photoaffinity label for acyl-CoA:glycine N-acyltransferase (EC 2.3.1.13) from beef liver. p-Azidobenzoyl-CoA is an active-site-directed reagent for this N-acyltransferase, since it is an alternate substrate (Km = 26 micronM, when [glycine] = 100 mM). Ultraviolet irradiation of a mixture of p-azidobenzoyl-CoA and the N-acyltransferase produces irreversible inhibition. Benzoyl-CoA protects the enzyme from inhibition by photoactivated p-azidobenzoyl-CoA. Acyl-CoA:glycine N-acyltransferase is composed of a single polypeptide with a molecular weight of about 35 000. Photolabeling experiments show that there is one active site per molecule of enzyme.
A photoaffinity analogue of ATP, 8-azido-adenosine 5′-triphosphate (8-N3ATP), was used to probe ATP-binding sites in native transcription complexes of vesicular stomatitis virus (VSV) (New Jersey serotype). The analogue was found to be a substrate for a serine/threonine protein kinase that phosphorylated both the NS and L proteins of native complexes. The analogue failed to interact with the RNA polymerase, another ATP-utilizing activity associated with the transcription complex. Kinetic analyses of both ATP and 8-N3ATP utilization by the protein kinase yielded biphasic saturation curves. Photolysis of 8-N3ATP in the presence of VSV transcription complexes resulted in selective labelling of the L protein. The photolabelling of L was saturable and apparently biphasic. Photolabelling of the L protein was significantly reduced by competition with ATP whereas other nucleoside triphosphates (GTP, UTP and CTP) were ineffective competitors. The stoichiometry of photolabelling was 0.2 at 10 µm-8N3ATP and 1.3 at 100 µm-ATP. These data provide chemical evidence for a virus-encoded serine/threonine protein kinase which resides on the L protein.
In an effort to identify the polypeptide chain of glucosylphosphodolichol synthase (EC 2.4.1.117), yeast microsomal membranes were allowed to react with 5-azido[beta-32P]UDPGlc, a photoactive analogue of UDPGlc, which is a substrate for this enzyme. Upon photolysis the 32P-labeled probe was shown to link covalently to a 35-kDa protein present in microsomal membranes prepared from several wild-type yeast strains. Binding was either reduced or absent in the microsomal membranes from two yeast mutants (alg5 and dpg1) that are known to be defective in the synthesis of glucosylphosphodolichol. The microsomes isolated from a heterozygous diploid strain alg5::dpg1 generated from these two mutants exhibited partial restoration of both the ability to photolabel the 35-kDa protein and the ability to catalyze the synthesis of glucosylphosphodolichol. Microsomal membranes from a mutant strain that synthesized glucosylphosphodolichol but lacked the ability to transfer the glucosyl residue to the growing lipid-linked oligosaccharide (alg6) exhibited labeling with 5-azido[beta-32P]UDPGlc comparable to that found in microsomes from the wild-type strain. In all cases photoinsertion of the probe into the 35-kDa protein correlated with the level of synthase assayed in the microsomal membranes. These results strongly support the conclusion that the 35-kDa protein labeled in these experiments is a component of glucosylphosphodolichol synthase.
A photoactive coenzyme analog of NAD+ has been synthesized by chemically coupling [32P]2-azido-AMP and NMN to produce [32P]nicotinamide 2-azidoadenosine dinucleotide (2-azido-NAD+). The utility of 2-azido-NAD+ as an effective active-site-directed photoprobe was demonstrated using bovine liver glutamate dehydrogenase as a model enzyme. In the absence of ultraviolet light, 2-azido-NAD+ is a substrate for this enzyme. Photoincorporation of probe was saturable with two different apparent dissociation constants of 10 microM and 40 microM. Protection of photoinsertion was seen with the natural substrate NAD+ with apparent dissociation constants of less than 5 microM and 25 microM. This observation may be explained on the basis of negative cooperative interaction between the subunits. The photoinsertion of 2-azido-NAD+ was increased by GTP and decreased by ADP in accordance with their known effects on NAD+ binding. When the enzyme was covalently modified by photolysis in the presence of saturating amounts of photoprobe, an approximately 40% inhibition of the enzyme activity was observed. These results demonstrate that the photoaffinity coenzyme analog has potential application as a probe to characterize NAD(+)-binding proteins and to identify the active sites of these proteins.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFormation of an intramolecular cystine disulfide during the reaction of 8-azidoguanosine 5'-triphosphate with cytosolic phosphoenolpyruvate carboxykinase (GTP) causes inactivation without photolabelingCristina T. Lewis, Boyd E. Haley, and Gerald M. CarlsonCite this: Biochemistry 1989, 28, 24, 9248–9255Publication Date (Print):November 1, 1989Publication History Published online1 May 2002Published inissue 1 November 1989https://pubs.acs.org/doi/10.1021/bi00450a003https://doi.org/10.1021/bi00450a003research-articleACS PublicationsRequest reuse permissionsArticle Views61Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
The photoaffinity probe 5-azido-uridine 5{prime}-{beta}-({sup 32}P)-diphosphate glucose was used to identify the major UDPG-binding polypeptide of red beet (1,3)-{beta}-glucan synthase. Glucan synthase was purified from plasma membranes by sequential solubilization with CHAPS followed by product entrapment. Two major polypeptides at 72 and 54 kD were labelled by probe. Labelling of both was abolished with increasing levels of cold UDPG. However, labelling of the 54 kD polypeptide was dependent upon the presence of divalent cations. These data suggest that the 54 kD polypeptide is a substrate-binding and cation-regulated component of the glucan synthase complex.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT2- and 8-Azido photoaffinity probes. 2. Studies on the binding process of 2-5A synthetase by photosensitive ATP analogsRobert J. Suhadolnik, Shi Wu Li, Robert W. Sobol, Jr., and Boyd E. HaleyCite this: Biochemistry 1988, 27, 24, 8846–8851Publication Date (Print):November 1, 1988Publication History Published online1 May 2002Published inissue 1 November 1988https://pubs.acs.org/doi/10.1021/bi00424a024https://doi.org/10.1021/bi00424a024research-articleACS PublicationsRequest reuse permissionsArticle Views61Altmetric-Citations9LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate‐binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [γ‐ 32 P]GTPγBP and [γ‐ 32 P]ATPγBP. Without activating light, GTPγBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPγBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [γ‐ 32 P]GTPγBP and [γ‐ 32 P]ATPγBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe 3+ ‐chelate affinity chromatography and reversed‐phase HPLC, photolabeled peptides located within or near the phosphate‐binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPγBP primarily modified a peptide near the middle of the GDH sequence, Asn135–Lys143 and Glu290–Lys295. However, ATPγBP modified a single peptide corresponding to the sequence Met411–Arg419 near the C‐terminal domain. Using these results and the data from the previously identified base‐binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three‐dimensional schematic model structure derived from the bacterial enzyme.
