Characterization of the substrate-binding sites of the mitochondrial nicotinamide nucleotide transhydrogenase

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is modified and inhibited by p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The modification appears to occur at the NAD(H)-binding site when TH alone or TH in the presence of NADPH is incubated with FSBA. However, when this site is protected by NADH, then FSBA inhibits TH more slowly and modifies a different, though specific, site. This second site could be the NADP(H)-binding site. Using [3H]FSBA in the presence of NADPH, the NAD(H)-binding site was modified, and a single tryptic peptide carrying the label was isolated and sequenced. The amino acid sequence of this peptide was Glu-Ser-Gly-Glu-Gly-Gln-Gly-Gly-Tyr*-Ala-Lys. The modified residue was Tyr. The labeled peptide isolated after incubating TH with [3H]FSBA in the presence of NADH could not be completely purified. However, amino acid analysis and partial sequencing made it possible to identify this segment on the amino acid sequence of bovine TH as derived from its cDNA by Yamaguchi et al. (private communication).     

Effects of N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline on hydride ion transfer and proton translocation activities of mitochondrial nicotinamidenucleotide transhydrogenase

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the mitochondrial energy-linked nicotinamidenucleotide transhydrogenase (TH). Our studies [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221; Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480] suggested that the inhibition site of DCCD is near the NAD(H) binding site, because NAD(H) and competitive inhibitors protected TH against inhibition by DCCD and, unlike the unmodified TH, the DCCD-modified TH did not bind to NAD-agarose. Others [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] could not demonstrate protection by NADH, obtained data indicating DCCD inhibits proton translocation by TH much more than hydride ion transfer from NADPH to 3-acetylpyridine adenine dinucleotide (AcPyAD), and concluded that DCCD modifies an essential residue in the proton channel of TH. The present studies show that N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inhibits TH. The inhibition is pseudo first order at several EEDQ concentrations, and the reaction order with respect to [EEDQ] is unity, suggesting that inhibition involves the interaction of one molecule of EEDQ with one active unit of TH. The EEDQ-modified TH reacts covalently with [3H]aniline, suggesting that the residue modified by EEDQ is a carboxyl group. More significantly, it has been shown that the absorbance change of oxonol VI at 630 minus 603 nm is a reliable reporter of TH-induced membrane potential formation in submitochondrial particles and that TH-catalyzed hydride ion transfer from NADPH to AcPyAD and the membrane potential induced by this reaction are inhibited in parallel by either DCCD or EEDQ.     

Mitochondrial nicotinamide nucleotide transhydrogenase: nonidentical modification by N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline at the NAD(H) binding site

The energy-linked nicotinamide nucleotide transhydrogenase (TH) purified from bovine heart mitochondria is inhibited by the carboxyl group modifiers, N,N'-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ). With either reagent, complete activity inhibition corresponds to modification of one carboxyl group per 2 mol (monomers) of this dimeric enzyme, suggesting half-site reactivity toward DCCD and EEDQ [D. C. Phelps, and Y. Hatefi (1984) Biochemistry 23, 4475-4480; 6340-6344]. It has also been shown in the former reference that DCCD appears to modify TH at the NAD(H)-binding site. The present paper presents data suggesting that EEDQ also binds at or near the NAD(H)-binding domain of TH, but at a site not identical to that of DCCD: TH modified with and inhibited approximately 85% by EEDQ could be further labeled with [14C]DCCD to the extent of 70% of the maximum in the same time period that unmodified TH was modified by [14C]DCCD to near saturation (1 mol DCCD/TH dimer); DCCD-modified TH did not bind to NAD-agarose, while EEDQ-modified TH showed partial affinity for NAD-agarose; 5'-AMP completely protected TH against modification by DCCD, but showed only a weak protective effect against EEDQ; by contrast, NMNH, which is a TH substrate and binds to the NADH site, did not protect TH against DCCD, but completely protected the enzyme against attack by EEDQ. The results are consistent with the possibility that DCCD modifies TH where the 5'-AMP moiety of NAD(H) binds, while EEDQ modifies the enzyme where the NMN(H) moiety of NAD(H) resides.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.     

Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking

Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site [Abraham, K. I., & Modak, M. J. (1984) Biochemistry 23, 1176-1182], we have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with [alpha-32P]dTTP. Covalent cross-linking of [alpha-32P]dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria: (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the [alpha-32P]dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these smaller peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. We concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.     

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.     

ADP ribosylation of rat liver lysine-rich histone in vitro.

Purified rat liver nuclei were incubated with [14C]-NAD+ and the various nuclear protein fractions were separated. Forty per cent of the total radioactivity incorporated was associated with the histone fraction. Of this, about 50% was extracted with H1, in 0.5 N perchloric acid. When crude H1 was purified and fractionated into five different subfractions by chromatography on Bio-Rex 70, it was found that all the H1 subfractions contained radioactivity. This radioactive material was identified as oligomers of adenosine diphosphate ribose (ADP-Rib) with an average chain length which corresponded to trimers. The extent of the modification was dependent on the concentration of NAD+. About 60% of the H1 molecules were modified with a concentration of 1 mM NAD+. The presence of these oligomers of ADP-Rib introduced a large degree of microheterogeneity to H1 as detected by electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid. Bands of H1 with 10 to 20% less mobility than the unmodified H1 were present. Also, as a consequence of large content of ADP-Rib, the absorption maximum shifted from 275 to 259 nm. The half-life of the bond between the oligomers of ADP-Rib and H1 was about 3 min at 37 degrees C in the presence of 0.1 N NaOH, and 10 m1 were modified. The site of ADP ribosylation in the NH2-terminal half was localized in the tryptic peptide extending from the NH2-terminal end to lysine 15. The site of modification of the COOH-erminal half was localized in the tryptic peptide which contained the only glutamic acid residue in this fragment of H1...     

Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.     

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.     

Amino acid sequence of the nucleotide-binding site of D-(-)-beta-hydroxybutyrate dehydrogenase labeled with arylazido-beta-[3-3H]alanylnicotinamide adenine dinucleotide

In the dark, arylazido-beta-alanylnicotinamide adenine dinucleotide (N3-NAD) can replace NAD as cofactor for D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria. When photoirradiated with visible light, N3-NAD forms a nitrene species that binds covalently to BDH and inhibits the enzyme. NAD(H) protects BDH against photolabeling and inhibition by N3-NAD [Yamaguchi, M., Chen, S., & Hatefi, Y. (1985) Biochemistry 24, 4912-4916]. In the present study, a tryptic peptide of purified BDH photolabeled with arylazido-beta-[3-3H] alanyl-NAD [( 3H]N3-NAD) was isolated and sequenced. The same tryptic peptide was also isolated from BDH not labeled with [3H]N3-NAD and sequenced. Both peptides indicated the sequence Met-Glu-Ser-Tyr-Cys-Thr-Ser-Gly-Ser-Thr-Asp-Thr-Ser-Pro-Val-Ile-Lys. The residue labeled with [3H]N3-NAD was Cys. This heptadecapeptide contains 14 uncharged residues and is marked by having in an undecapeptide segment 8 hydroxy amino acids located symmetrically around a central glycine.     

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).     

The mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species

A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.     

Identification of the arylazido-beta-alanyl-NAD+-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry

We have identified the site labeled by arylazido-beta-alanyl-NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+) in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry. This NAD+ photoaffinity analogue has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in a very specific manner and probably at the active site of the enzyme [Chen, S., Davis, H., Vierra, J. R., & Guillory, R. J. (1984) Biochem. Biophys. Stud. Proteins Nucleic Acids, Proc. Int. Symp., 3rd, 407-425]. The label is associated exclusively with a tryptic peptide that has the sequence Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue at position seven was predominantly labeled and suggested to be the site modified by arylazido-beta-alanyl-NAD+. This cysteine residue corresponds to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity. The present investigation clearly demonstrates that arylazido-beta-alanyl-NAD+ is a useful photoaffinity probe to characterize the active sites of NAD(H)-dependent enzymes.     

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.     

Reduction of the beta-Cys-gamma-Glu thiol ester bond of human C3 with sodium borohydride

Further chemical evidence has been obtained using NaB3H4 to support our previous assignment of a thiol ester bond in human C3 (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768). Following trypsin activation of human C3 in the presence of NaB3H4, 3H was shown to have incorporated specifically into the alpha'-chain of C3b. Subsequent fragmentation of [3H]C3b with porcine elastase further localized the label to the C3d subdomain. Under identical conditions, native C3 or C3 pretreated with trypsin (C3b) showed low reactivity with NaB3H4. A tryptic peptide containing the 3H label was isolated following digestion of [3H]C3b on activated thiol-Sepharose. After hydrolysis and saponification of the peptide hydrolysate, amino acid analysis indicated that the 3H had been incorporated into alpha-amino-delta-hydroxyvaleric acid, the product expected from reduction of an ester bond involving a glutamyl residue. On sequence analysis of the labeled peptide, the 3H was shown to reside at the position of the glutamyl residue previously proposed to be involved in the thiol ester bond. The residue at this position was confirmed as alpha-amino-delta-[3H] hydroxyvaleric acid by high performance liquid chromatography analysis and, after back hydrolysis, by amino acid analysis. These data significantly strengthen earlier studies which indicated the presence of a beta-Cys-gamma-Glu thiol ester bond in human C3.