Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose-incorporated peptides

A photoaffinity substrate analogue, 8-azido-ADP-[14C]glucose, reacts specifically and covalently with Escherichia coli ADP-glucose synthetase. The site(s) of reaction of 8-azido-ADP-[14C]glucose with the enzyme was identified by isolation of tryptic peptides containing the labeled analogue by use of high performance liquid chromatography technique and subsequent NH2-terminal sequence analysis of the purified radioactive peptides. One major binding region of the azido analogue is a peptide segment composed of residues 107-114 of the enzyme's polypeptide chain. Lys 108 and Arg 114 become trypsin-resistant sites when the enzyme is photoinactivated by 8-azido-ADP-[14C] glucose, suggesting that the analogue binds at or near the vicinity of these 2 basic amino acid residues. Conformational analysis of this peptide segment (residues 107-114) shows a strong probability of a reverse beta-turn secondary structure, suggesting that this peptide segment is on the enzyme surface. Two minor reaction regions of the enzyme with the analogue were also identified by chemical characterization. One region was composed of residues 162-207. Lys 194 was previously suggested as the activator-binding site by chemical modification studies with pyridoxal phosphate (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). Another minor region where the analogue binds the tryptic peptide composed of residues 380-385 is near the COOH-terminal side of the enzyme. It is postulated that all these peptide segments are juxtaposed in tertiary structure.     

Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

A pterin analogue, 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone (ANBADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a Ki of 8.8 +/- 1.1 microM. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. Phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87 +/- 0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from [3H]ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with [3H]ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. The reactive sulfhydryl of phenylalanine hydroxylase associated with activation of the enzyme was also identified by labeling with the chromophoric label 5-(iodoacetamido)fluorescein [Parniak, M. A., & Kaufman, S. (1981) J. Biol. Chem. 256, 6876]. Labeling of the enzyme resulted in 1 mol of fluorescein bound per phenylalanine hydroxylase subunit and a concomitant activation of phenylalanine hydroxylase to 82% of the activity found with phenylalanine-activated enzyme. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.     

The interaction site for tamoxifen aziridine with the bovine estrogen receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.     

Total number and differentiation of nucleotide binding sites on mitochondrial F1-ATPase. An approach by photolabeling and equilibrium binding studies

In this study 3'-O-[3-(4-azido-2-nitrophenyl)propionyl]-ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide-depleted F1-ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki = 50 microM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between alpha and beta subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3'-ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows. The total number of sites which can be simultaneously occupied by nucleotides on F1 is six. Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727] that the high-affinity sites are the catalytic ones which can be covalently labeled by 3'-O-[5-azidonaphthoyl(1)]-ADP [Lübben, M., Lücken, U., Weber, J. & Sch?fer, G. (1984) Eur. J. Biochem. 143, 483-490], it appears likely that azidonitrophenylpropionyl-ADP is a specific photolabel for the lower-affinity sites on nucleotide-depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs. The labeled low-affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.     

Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.     

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

The photoreactive AMP analog, 8-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 microM and 1.5 microM, 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. Photolabeling of AMP-free dehydratase with 8-azido-[2-3H]AMP resulted in a time and concentration-dependent enzyme inactivation and concomitant incorporation of 8-azido-AMP into protein. At low 8-azido-AMP concentrations, incorporation of about 1 mol 8-azido-AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8-azido-AMP binding. Ultraviolet irradiation with 20 microM 3H-labeled 8-azido-AMP revealed one tryptic peptide, Thr230-Thr-Gly-Thr-Leu-Ala-Asp-Gly-Cys-Asp-Val-Ser-Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8-azido-AMP, most likely represents the AMP-binding region on the dehydratase molecule.     

Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase

Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites (Modak, M. J., and Gillerman-Cox, E. (1982) J. Biol. Chem. 257, 15105-15109), we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.     

Tyrosine 264 in the recA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azidoadenosine 5'-triphosphate

The photoaffinity label 8-azidoadenosine 5'-triphosphate (N3-ATP) was used to covalently modify the recA protein from Escherichia coli within its ATP-binding site. We have previously demonstrated that N3-ATP modification of recA protein is specific for the ATP-binding site and have isolated a unique tryptic peptide (T31), spanning residues 257-280, that contains the exclusive site of attachment of this ATP analog (Knight, K. L., and McEntee, K. (1985) J. Biol. Chem. 260, 867-872). We performed a secondary proteolytic digestion of the [alpha-32P]N3-ATP-labeled T31 peptide using Staphylococcus aureus V8 protease and purified the resulting peptide fragments by high-pressure liquid chromatography (HPLC). Based on a comparison of the amino acid compositions of all purified fragments and sequence analysis of one labeled fragment we determined that Tyr-264 is the exclusive site of N3-ATP attachment in recA protein. Photoaffinity labeling of recA protein was also performed in the presence of single-stranded DNA. Following trypsin treatment and separation of peptides by HPLC we showed that tryptic peptide T31 contained the exclusive site of N3-ATP attachment. A secondary proteolytic digestion was performed on both [alpha-32P]N3ATP-modified T31 and unmodified T31 using alpha-chymotrypsin. Comparison of the HPLC profiles and amino acid compositions of the resulting fragments was consistent with Tyr-264 as the exclusive site of N3-ATP attachment to recA protein.     

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction among four residues distributed through the secretin pharmacophore and a focused region of the secretin receptor amino terminus

The amino terminus of the secretin receptor (SecR) is known to be critical for natural agonist action, although the role it plays is still unclear. We have demonstrated that photolabile residues within both the amino-terminal (position 6) and carboxyl-terminal (positions 22 and 26) halves of secretin each covalently label receptor amino-terminal tail residues [Dong et al., J Biol Chem, 274:19161-19167 (1999), 274:903-909 (1999), and 275:26032-26039 (2000)]. Here, we extend this series of studies with an additional probe having its site of covalent attachment in a distinct region of the peptide, between amino- and carboxyl-terminal helical domains. This probe incorporated a photolabile (epsilon-p-benzoylbenzoyl)lysine in position 18 and a site for oxidative radioiodination [(tyrosine(10),(benzoyl-benzoyl)lysine(18))rat secretin-27]. This analog represented a full agonist, stimulating cAMP accumulation in Chinese hamster ovary-SecR cells in a concentration-dependent manner. It bound to the SecR specifically and saturably, and was able to efficiently label that molecule within its amino terminus. Sequential specific cleavage, purification, and sequencing demonstrated that this probe labeled receptor residue arginine(14), in the same subdomain as that labeled by previous probes. Consistent with the importance of this residue, alanine replacement mutagenesis (R14A) resulted in substantial reductions in the potency (127-fold) and binding affinity (400-fold) of secretin relative to its action at the wild-type receptor. We have been able to accommodate all four extant pairs of residue-residue approximations between divergent regions of the secretin pharmacophore and the first forty residues of the SecR into a credible molecular model of this interaction. Additional experimentally derived constraints will be necessary to determine the spatial positioning of this complex with the remainder of the SecR.     

N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.     

Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line

The O-linked oligosaccharides of the cloned, murine cytotoxic T cell line B6.1.SF.1 were compared with the corresponding oligosaccharides from a Vicia villosa lectin-resistant mutant of B6.1.SF.1 called VV6 (Conzelmann, A., Pink, R., Acuto, O., Mach, J.-P., Dolivo, S., and Nabholz, M. (1980) Eur. J. Immunol. 10, 860-868). The VV6 mutant cells are deficient in binding sites for this GalNAc-specific lectin. Cells were grown in the presence of [3H]glucosamine and [3H] galactose to label the glycoproteins, and the desialyzed, alkaline borohydride-released oligosaccharides were isolated and characterized. The VV6 cells contained a series of O-linked oligosaccharides ranging in size from a disaccharide to a pentasaccharide. These were composed of galactose, N-acetylglucosamine, and N-acetylhexosaminitol, the latter sugar being derived from the reducing terminus. The predominant oligosaccharide had the partial structure Gal beta GlcNAc beta-(Gal beta)N-acetylhexosaminitol. In contrast, the analogous oligosaccharides of the parental cells contained additional beta-linked GalNAc residues located at nonreducing termini. The smallest of these had the structure GalNAc beta 1,4Gal beta-N-acetylhexosaminitol. Neither cell line contained significant amounts of terminal GalNAc linked to Ser/Thr which is the main binding site for the V. villosa B4 lectin on Tn erythrocytes (Tollefsen, S. R., and Kornfeld, R. (1983) J. Biol. Chem. 258, 5172-5176). These findings suggest that the major binding sites for the V. villosa lectin on the parental cytotoxic T cell line consist of structures containing beta 1,4-linked GalNAc residues at the nonreducing ends of conventional O-linked structures. The VV6 cells lack these beta-linked GalNAc residues, and this may account for their deficiency of V. villosa lectin-binding sites. In the following paper (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12536-12542), we demonstrate that the VV6 cells are missing the N-acetylgalactosaminyltransferase that is responsible for the synthesis of these unusual oligosaccharides.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Active site labeling of dopamine beta-hydroxylase by two mechanism-based inhibitors: 6-hydroxybenzofuran and phenylhydrazine

6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Inhibition of fructose-1,6-biphosphatase by the photoaffinity AMP analog, 8-azidoadenosine 5'-monophosphate.

Inhibition studies with the photoreactive AMP analog, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), demonstrate that this compound is, like AMP, an allosteric inhibitor of pig kidney and muscle fructose-1,6-biphosphateses. Photolysis of a mixture of purified pig kidney fructose-1,6-biphosphate and 8-azido-[14C]AMP results in the loss of enzyme activity and the reagent is incorporated to the protein. The incorporation of reagent linearly correlates with the loss of enzyme activity. Extrapolation to zero activity correlates with the incorporation of 3.7 mol of reagent/mol of enzyme (i.e. 0.9 per subunit). Thus, 8-azido-AMP appears to be a photoaffinity label for the allosteric AMP binding site of fructose-1,6-biphosphatase.     

A specific photoaffinity label for hemolymph and ovarian juvenile hormone-binding proteins in Leucophaea maderae

A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.     

Photoaffinity labeling of the tetrabenazine binding sites of bovine chromaffin granule membranes

An azido derivative of tetrabenazine, a specific inhibitor of the monoamine carrier of chromaffin granule membranes, has been synthesized. In the dark, this compound, 3H-labeled N-(3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo [a]quinolizin-2-yl)-4-[(4-azido-2-nitrophenyl)amino]butanamide+ ++ ([3H]TBA), bound reversibly to purified chromaffin granule membranes. Centrifugation through SP-Sephadex columns was used to separate bound and free [3H]TBA. This technique gave low levels of nonspecific binding and allowed recovery of [3H]TBA-membrane complexes. Scatchard analysis of the data indicated one class of sites with an equilibrium dissociation constant KD of 50 nM and a density of sites of 40-50 pmol/mg of protein, consistent with reported densities of reserpine and dihydrotetrabenazine binding sites. Competition experiments showed that TBA and tetrabenazine bound to the same site. Irradiation at 435 nm of [3H]TBA-membrane mixtures induced some irreversible binding of the probe to membranes. After irreversible binding of TBA, the number of dihydrotetrabenazine binding sites was decreased, indicating that the probe was covalently bound to the monoamine carrier. [3H]TBA-membrane complexes isolated by centrifugation through SP-Sephadex columns were irradiated, and their radioactivity was analyzed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. A polypeptide with a molecular weight of 70 000 was labeled. This polypeptide was different from dopamine beta-hydroxylase, and it was not adsorbed on concanavalin A-Sepharose. It is proposed that the monoamine carrier of chromaffin granule membrane has an oligomeric structure, involving a 45K subunit [Gabizon, R., Yetinson, T., & Schuldiner, S. (1982) J. Biol. Chem. 257, 15145] and a 70K subunit.     

Photoaffinity labeling of tubulin with (2-nitro-4-azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites

A new photoaffinity analogue of colchicine, (2-nitro-4-azidophenyl)deacetylcolchicine (NAPDAC), bound to two classes of sites on bovine renal tubulin and photolabeled both the alpha- and beta-subunits. The apparent Ki for the photoaffinity analogue was 1.40 +/- 0.17 microM (mean +/- SD, n = 3) as measured by competition with [3H] colchicine. Values of the apparent KdS for the two sites, as measured by the direct binding of the [3H]NAPDAC to tubulin, were 0.48 +/- 0.11 microM and 11.6 +/- 3.5 microM (mean +/- SD, n = 6), and the corresponding stoichiometries of binding of the two sites were 0.25 +/- 0.06 and 1.3 +/- 0.4 mol/mol of tubulin (mean +/- SD, n = 6). NAPDAC was a potent inhibitor of microtubule formation as detected by electron microscopy. When tubulin was photolabeled with NAPDAC at 25 degrees C, 15 +/- 3 mol % (mean +/- SD, n = 6) of the [3H]NAPDAC was covalently bound to the alpha-subunit, and 67 +/- 9 mol % (mean +/- SD, n = 6) was covalently bound to the beta-subunit. Since NAPDAC is a mixture of two interconvertible diastereomers, the photoincorporation of each was also examined. One diastereomer photolabeled both alpha- and beta-tubulin; however, the other did not significantly photolabel either subunit. Tubulin photolabeled with NAPDAC (1:1 mole ratio) exhibited a 23% decrease in colchicine binding. Preblocking and prephotolysis experiments with colchicine, NAPDAC, or ANPAH-CLC [Williams et al. (1985) J. Biol. Chem. 260, 13794-13802] provided evidence for conformational changes in tubulin upon colchicine binding. Peptide maps of [3H]NAPDAC-labeled alpha- and beta-tubulin, using Staphylococcus aureus V8 protease, demonstrated the presence of NAPDAC in one peptide of the alpha-subunit and in five peptides of the beta-subunit as detected by autoradiography. NAPDAC provides the first direct evidence for two colchicine binding sites on tubulin.     

Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis.

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630----Glu or Tyr632----Phe caused no effect on the enzyme activity, that of Tyr632----Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.