Direct photoaffinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu185 of the heavy chain at the active site

The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with [3H]UDP. [3H] UDP was stably trapped at the active site by addition of vanadate (Vi) and Co2+. The extraordinary stability of the myosin.Co2+.[3H]UDP.Vi complex (t1/2 greater than 5 days at 0 degrees C) allowed it to be purified free of extraneous [3H]UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped [3H]UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results (Maruta, H., and Korn, E. (1981) J. Biol. Chem. 256, 499-502) using [3H]UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a "zero-length" cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by [3H]UTP photolabeling in Acanthamoeba myosin II (Atkinson, M. A., Robinson, E. A., Appella, E., and Korn, E. D. (1986) J. Biol. Chem. 261, 1844-1848).     

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serine-324 of myosin's heavy chain is photoaffinity-labeled by 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate

A portion of the active site of rabbit skeletal myosin near the ribose ring of ATP can be labeled by the photoaffinity analogue 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate (Bz2ATP). The specificity of the photolabeling was assured by first trapping [14C]Bz2ATP at the active site by use of thiol cross-linking agents [Mahmood, R., Cremo, C., Nakamaye, K., & Yount, R. (1987) J. Biol. Chem. 262, 14479-14486]. Five radioactive peptides were isolated by high-performance liquid chromatography after extensive trypsin and subtilisin digestion of photolabeled myosin subfragment 1. Four of these peptides were sequenced by Edman techniques, and all originated from a region with the sequence Gly-Glu-Ile-Thr-Val-Pro-Ser-Ile-Asp-Asp-Gln, which corresponds to rabbit myosin heavy chain residues 318-328. The fifth labeled peptide had an amino acid composition appropriate for residues 312-328. Amino acid composition, radiochemical analysis, and sequence data indicate that Ser-324 is the major amino acid residue photolabeled by Bz2ATP. Spectrophotometric evidence indicates that the benzophenone carbonyl group has inserted into a C-H bond from either the alpha- or beta-carbon of serine. These results place Ser-324 at a distance of 6-7 A from the 3'(2') ribose oxygens of ATP bound at the active site of myosin.     

Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.     

Transmission of ADP.vanadate-induced conformational changes to three peptide segments of myosin subfragment-1

In order to study the conformational changes associated with formation of the stable ternary complex of myosin subfragment-1 (S-1) with ADP and orthovanadate (Vi), S-1 was fluorescently labeled with 9-anthroylnitrile, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and 5-(iodoacetamido) fluorescein at the 23-, 50-, and 20-kDa peptide segments of S-1, respectively (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194; Hiratsuka, T. (1986) J. Biol. Chem. 261, 7294-7299; Takashi, R. (1979) Biochemistry 18, 5164-5169). The extrinsic fluorescence of these S-1 derivatives was sensitive not only to binding of ADP but to formation of the stable ternary complex with ADP and Vi. By using these fluorescent properties, the kinetics of formation of the stable ternary complexes of these S-1 derivatives with ADP and Vi, M. ADP.Vi, were analyzed according to the scheme proposed by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2620-2624). [Formula; see text] The values obtained for KVi )0.2-0.4 mM) and k (0.03-0.05 s-1) of these S-1 derivatives were similar regardless of the peptide segments of S-1 where the fluorophore had been covalently labeled. These results suggest that the conformational changes, which are induced by formation of the stable ternary complex of S-1 with ADP and Vi, are transmitted to all three peptide segments of S-1 at a similar rate. The present results also encourage us to confirm that the ATPase site of S-1 resides at or near the region where all three peptide segments of S-1 are contiguous.     

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.     

UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.     

The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate

The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.     

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.     

Direct chemical evidence that serine 180 in the glycine-rich loop of myosin binds to ATP

The photochemical reaction of MgADP-vanadate with the active site of myosin has been used to place a serine at the binding site for the gamma-phosphate of ATP. Irradiation of the MgADP-vanadate myosin subfragment 1 transition state-like complex with UV light specifically photooxidizes the hydroxyl group of a serine residue to an aldehyde (Cremo, C. R., Grammer, J. C., and Yount, R. G. (1988) Biochemistry 27, 8415-8420). Reduction of photooxidized myosin with Na-B3H4 gave only 3H-labeled serine. Here, subsequent extensive proteolytic digestion of 3H-labeled myosin subfragment 1 with trypsin and thermolysin yielded two 3H-labeled peptides, both of which contained the sequence Gly-Glu-Ser-Gly-Ala-Gly-Lys-Thr, in which all the 3H was associated with the serine. This sequence is conserved in all myosin heavy chains sequenced to date and corresponds to residues 178-185 in the rabbit myosin heavy chain (Tong, S. W., and Elzinga, M. (1983) J. Biol. Chem. 21, 13100-13110). These results place Ser-180 at the gamma-phosphate-binding site for ATP and indicate that the glycine-rich loop around the serine provides essential elements of the phosphate-binding site for ATP in all myosin molecules. Such a role was previously suggested based on the common sequence Gly-X-X-X-X-Gly-Lys-Thr/Ser, found in myosin and many other nucleotide-binding enzymes (Walker, J. E., Saraste, M., Runswick, M. H., and Gay, N. J. (1982) EMBO J. 1, 945-951).     

Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-AMP-incorporated peptides

The photoaffinity inhibitor analog [2-3H]8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase. The reaction site(s) of [2-3H]8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog. Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label. The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity. A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region. This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-[14C]glucose (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido-AMP were also identified by chemical characterization. One region, containing 20% of the covalently bound label, was composed of residues 11-68. This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label. The three modified peptide regions may be juxtaposed in the enzyme's 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.     

Amino acid sequence of the active site of Acanthamoeba myosin II

We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.     

Identification of Gln726 in nidogen as the amine acceptor in transglutaminase-catalyzed cross-linking of laminin-nidogen complexes.

The laminin-nidogen complex, the most abundant noncollagenous component of basement membranes, was recently shown to be a specific substrate for tissue transglutaminase (Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317). Saturation experiments to determine the number of amine acceptor site(s) indicated a single reactive Gln residue in nidogen and none in laminin. Murine nidogen was labeled with [3H]putrescine in the tissue transglutaminase-catalyzed reaction, and two major radioactively labeled fragments, T70 and T40, were isolated after limited trypsin digestion. NH2-terminal sequencing showed that T40 is contained in T70 and corresponds to the rodlike structure of nidogen, made up of epidermal growth factor-like repeats. Three radioactively labeled peptides, obtained by extensive trypsin digestion of reduced and alkylated T40, were sequenced. In all a single residue, Gln726, was found to contain label. Sequencing of additional peptides, obtained after further treatment of the largest radioactively labeled peptide with endoproteinase Asp-N, gave the same result. Gln726 is located in an exposed loop between the second and the third EGF-like repeat in nidogen. This site is also conserved in the human sequence.     

Characterization and function of catalytic subunit alpha of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. A study with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Subunit alpha (Mr 89,000) from vacuolar membrane H+-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae was found to bind 8-azido[alpha-32P]adenosine triphosphate. Labeling by this photosensitive ATP derivative was saturable with an apparent dissociation constant of 10(-6) to 10(-5) M and decreased in the presence of ATP and ADP. The enzyme was inactivated by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), with about 1 microM causing half-maximal inactivation in the neutral pH range. This inactivation was prevented by the presence of ATP, ADP, or adenosyl-5'-yl imidodiphosphate (AMP-PNP). The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol. Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme. When the enzyme was inactivated with [14C]NBD-Cl, subunit alpha was specifically labeled, and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP. From these results, it was concluded that subunit alpha of yeast vacuolar H+-ATPase has a catalytic site that contains a single, essential tyrosine residue. The kinetics of single site hydrolysis of [gamma-32P]ATP (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100) indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site. NBD-Cl inactivated the single site hydrolysis and inhibited the formation of an enzyme-ATP complex. Dicyclohexylcarbodiimide did not affect the single site hydrolysis, but inhibited the enzyme activity under steady-state conditions.     

Inhibition of muscle force by vanadate.

Vanadate (Vi), an analogue of inorganic phosphate (Pi), is known to bind tightly with a long half life to the myosin MgATPase site, producing a complex which inhibits force. Both of these ligands bind to an actin.myosin.ADP state that follows the release of Pi in the enzymatic cycle, and their effects on muscle fibers and proteins in solution provide information on the properties of this state. The inhibition of active force generation began to occur at a [Vi] of 5 microM and was 90% complete at a [Vi] of 1 mM. Hill plots of the inhibition of force by Vi approximated that expected for a simple binding isotherm. Similar plots were obtained at both 25 degrees C and 5 degrees C. A simple binding isotherm is not expected to occur in a muscle fiber where steric constraints imposed by the intact filaments should introduce more complexity into the energetics of ligand binding. The inhibition of MgATPase activity for acto-subfragment-1 to 50% of controls occurred at a [Vi] which was only 20-fold higher than that required to inhibit force generation in fibers to the same level. Some models of actomyosin interactions would predict that the range of [Vi] required for complete force inhibition in fibers and the difference in the [Vi] required for inhibition in fibers and of myosin in solution would both be much larger.     

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. [3H]meproadifen mustard reacts selectively with alpha-subunit Glu-262.

[3H]Meproadifen mustard, an affinity label for the noncompetitive antagonist site of the nicotinic acetylcholine receptor (AChR), specifically alkylates the AChR alpha-subunit when the acetylcholine-binding sites are occupied by agonist (Dreyer, E. B., Hasan, F., Cohen, S. G., and Cohen, J. B. (1986) J. Biol. Chem. 261, 13727-13734). In this report, we identify the site of alkylation within the alpha-subunit as Glu-262. AChR-rich membranes from Torpedo californica electric organ were reacted with [3H]meproadifen mustard in the presence of carbamylcholine and in the absence or presence of nonradioactive meproadifen to define specific alkylation of the noncompetitive antagonist site. Alkylated alpha-subunits were isolated and subjected to chemical or enzymatic cleavage. When digests with CNBr in 70% trifluoroacetic acid or 70% formic acid were fractionated by gel filtration high performance liquid chromatography (HPLC), specifically labeled material was recovered in the void volume fractions. Based upon NH2-terminal sequence analysis, for both digests, the void volume fractions contained a fragment beginning at Gln-208 before the M1 hydrophobic sequence, whereas the sample from the digest in trifluoroacetic acid also contained as a primary sequence a fragment beginning at Thr-244 and extending through the M2 hydrophobic sequence. Sequence analysis revealed no release of 3H for the sample from digestion in formic acid, whereas for the trifluoroacetic acid digest, there was specific release of 3H in cycle 19, which would correspond to Glu-262. This site of alkylation was confirmed by isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase HPLC of a specifically labeled fragment from an endoproteinase Lys-C digest of the alkylated alpha-subunit. NH2-terminal amino acid sequencing revealed release of 3H at cycle 20 from a fragment beginning at Met-243 and extending into the M3 hydrophobic sequence. Because [3H]meproadifen mustard contains, as its reactive group, a positively charged quaternary aziridinium ion, Glu-262 of the alpha-subunit is identified as a contributor to the cation-binding domain of the noncompetitive antagonist-binding site and thus of the ion channel.     

The interaction of Phe472 with a fluorescent inhibitor bound to the complex of myosin subfragment-1 with nucleotide

The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA) acts as a fluorescent inhibitor for the ATPases of skeletal [Hiratsuka (1994) J. Biol. Chem. 269, 27251-27257] and Dictyostelium discoideum [Bobkov et al. (1997) J. Muscle Res. Cell Motil. 18, 563-571] myosins. The former paper suggested that, upon addition of excess nucleotides to the binary complex of subfragment-1 from skeletal myosin (S1) with PPBA, a stable ternary complex of S1 with PPBA and nucleotide is formed. Useful fluorescence properties of PPBA enable us to distinguish the conformation of the myosin ATPase at the ATP state from that at the ADP state. In the present paper, to determine the PPBA-binding site in the complexes, enzymatic and fluorescence properties of the S1.PPBA.nucleotide complexes were investigated. Upon formation of the ternary complex with ATP, a new peak appeared at 398 nm in the PPBA fluorescence spectrum. Experiments using model compounds of aromatic amino acid suggested that this fluorescence peak at 398 nm is originated from PPBA interacting with Phe residue(s). Taking into account differences in fluorescence spectra between complexes of S1 and those of subfragment-1 from D. discoideum myosin (S1dC), in the ternary complex of S1 formed with ATP, PPBA was suggested to interact with Phe residue(s) that is absent in S1dC. Docking simulation of PPBA on the S1.nucleotide complex revealed that Phe472 interacts with PPBA. Binding sites of PPBA and blebbistatin, an inhibitor showing high affinity and selectivity toward myosin II [Kovács et al. (2004) J. Biol. Chem. 279, 35557-35563], seem to overlap at least partly.     

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.