Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.     

Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.

S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3', 5'-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.     

Effect of NAD on flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Flounder muscle (Pseudopleuronectes americanus) glyceraldehyde-3-phosphate dehydrogenase was characterized as to its stability towards various inactivating treatments in the presence and absence of the enzyme cofactor, NAD. Incubation of a partially purified enzyme preparation at urea concentrations greater than 2 M produced a very rapid inactivation. NAD greatly reduced the rate of inactivation at all the urea concentrations tested. Incubation of each of the three major muscle enzyme forms in 0.1 percent trypsin or chymotrypsin for forty-five minutes decreased the activity of each form by 65 percent and 55 percent, respectively. NAD (5mM) afforded complete protection to each enzyme form from proteolytic digestion by these two enzymes. Exposure of each form to 50 degrees or 20 mM ATP also led to gross inactivation which could be greatly reduced if the respective incubations were performed in the presence of 5mM NAD. NAD was also found to be required for the renaturation of the unfolded urea-denatured subunits to form the active tetramer.     

A single mutation at Tyr143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and affects the oxidation state of bound cofactor nicotinamide-adenine dinucleotide

Recently, we have described the first human case of AdoHcyase (S-adenosylhomocysteine hydrolase) deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) and the truncation mutation p.W112stop (AdoHcyase in which Trp112 is replaced by opal stop codon) were identified [Bari?, Fumi?, Glenn, Cuk, Schulze, Finkelstein, James, Mejaski-Bosnjak, Pazanin, Pogribny et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 4234-4239]. To elucidate the molecular and catalytic properties of AdoHcyase, we have made recombinant wild-type and mutant p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased from 65% to 75%. Further, the oxidation states of coenzyme NAD differ between mutant and wild-type protein, with an increased NADH accumulation in the mutant p.Y143C enzyme of 88% NADH (wild-type contains 18% NADH). Quantitative binding of NAD is not affected. Native polyacrylamide gel electrophoresis showed, that mutant p.Y143C subunits are able to form the tetrameric complex as is the wild-type enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7 degrees C compared with wild-type protein. Change of Glu115 to lysine in wild-type protein causes a change in thermosensitivity almost identical with that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Tyr143 and Glu115. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the AdoHcyase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with AdoHcyase deficiency.     

Mapping of the adenosine 5'-triphosphate binding site of type II calmodulin-dependent protein kinase

The specificity of the ATP-binding site of the type II calmodulin-dependent protein kinase was probed with 25 analogues of ATP modified at various positions of the molecule. The analogues were compared by their ability to compete with ATP in the protein kinase reaction. The result of this comparison indicates that the enzyme is most sensitive to modifications at, or replacement of, the purine moiety. Changes at the triphosphate chain are much better tolerated, although the enzyme exhibited a selective sensitivity to changes in the conformation of this group. The smallest contribution to the specificity of ATP binding appears to be made by the ribose ring. The Ki values obtained for a subset of these analogues were compared to those previously reported for phosphorylase b kinase and the cyclic nucleotide dependent protein kinases [Flockhart, D. A., Freist, W., Hoppe, J., Lincoln, T. M., & Corbin, J. D. (1984) Eur. J. Biochem. 140, 289-295]. A striking similarity in the responses of these protein kinases to modifications of the ATP molecule suggests that the type II calmodulin-dependent protein kinase is related to these enzymes. Support for this conclusion was provided, recently, through comparisons of the deduced primary structures of the alpha and beta subunits of the type II calmodulin-dependent protein kinase with the protein sequences of the catalytic subunits of phosphorylase b kinase and cAMP-dependent protein kinase [Hanley, R. M., Means, A. R., Ono, T., Kemp, B. E., Burgin, K. E., Waxham, N., & Kelly, P. T. (1987) Science (Washington, D.C.) 237, 293-297; Bennett, M. K., & Kennedy, M. B. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1794-1798], which indicated areas of extensive homology.     

Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. Evidence from methionine sulfoximine inhibition studies

Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme. Meister and co-workers (Manning, J. M., Moore, S., Rowe, W. B., and Meister, A. (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate. Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme. The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.     

Inhibition of S-adenosylhomocysteine hydrolase by acyclic sugar adenosine analogue D-eritadenine. Crystal structure of S-adenosylhomocysteine hydrolase complexed with D-eritadenine

D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase). Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors. Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase. The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues. The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+). However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD. The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes. The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues. The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+). By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction. DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor. On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.     

S-Adenosylhomocysteine hydrolase (AdoHcyase) deficiency: enzymatic capabilities of human AdoHcyase are highly effected by changes to codon 89 and its surrounding residues

Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Bari?, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538-545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Beluzi?, M. Cuk, T. Pavkov, K. Fumi?, I. Bari?, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245-253]. Now we report that the A89V exchange leads to a 70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5 degrees C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. 'Bioinformatics' analysis indicates that Val(89) collides with Thr(84) causing sterical incompatibility. Performing site-directed mutagenesis changing Thr(84) to 'smaller' Ser(84) but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr(84) with Lys(84) or Gln(84), thereby introducing residues with higher volume in proximity to Ala(89) results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.     

Regulation of CFTR Cl- channel gating by ADP and ATP analogues

The cystic fibrosis gene product (CFTR) is a chloride channel which, once phosphorylated, is regulated by nucleotide phosphates (Anderson, M. P., and M. J. Welsh. 1992. Science. 257:1701-1704; Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. 1994. Journal of General Physiology. 104:123-146). Nucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFTR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 +/- 9 microM. AMP neither initiated CFTR channel gating nor inhibited ATP-dependent CFTR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFTR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFTR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di- or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25 degrees C, result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.     

The ATP,Mg-dependent protein phosphatase. Regulation by inhibitor-1 or modulator protein and stabilizing role of Mg2+ ions

The activation of the ATP,Mg-dependent protein phosphatase [Fc.M] has been shown to involve a transient phosphorylation of the modulator subunit (M) and consequent isomerization of the catalytic subunit (Fc) into its active conformation (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S. -D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870). The modulator subunit constitutes the inactivating force for the enzyme, but the slow intramolecular inactivation of the phosphatase can be prevented or blocked by the addition of either the phosphorylated inhibitor-1 or Mg2+ ions. Autodephosphorylation of the modulator subunit is not prevented by the phosphoinhibitor-1, suggesting that the ATP,Mg-dependent phosphatase binds the phosphomodulator subunit in a very specific manner, different from the way it binds exogenous phosphoprotein substrates. Alternatively, the autodephosphorylation of the modulator subunit is catalyzed at a separate active site on the enzyme, which is not influenced by the binding of phosphoinhibitor-1. The phosphoinhibitor-1 does not prevent the activation of the enzyme by kinase FA when added at concentrations that totally inhibit the potential phosphorylase phosphatase activity. These results, together with other already published information, suggest separate autonomic controls of the ATP,Mg-dependent phosphatase activity by inhibitor-1 and the modulator protein through the presence of specific regulatory subunits on the enzyme.     

Cysteinyl peptide labeled by 3-bromo-2-ketoglutarate in the active site of pig heart NAD+-dependent isocitrate dehydrogenase

The substrate affinity label 3-bromo-2-ketoglutarate (BrKG) reacts covalently with pig heart NAD+-specific isocitrate dehydrogenase with complete inactivation and incorporation of about 0.8 mol of reagent/mol of average enzyme subunit [Bednar, R.A., Hartman, F.C., & Colman, R.F. (1982) Biochemistry 21, 3681-3689]. Protection against inactivation is provided by isocitrate and Mn2+. We have now identified a critical modified peptide by comparison of the peptides labeled by BrKG at pH 6.1 in the absence and presence of isocitrate and Mn2+. Modified enzyme, isolated from unreacted BrKG, was incubated with [3H]NaBH4 to reduce the keto group of protein-bound 2-ketoglutarate and thereby introduce a radioactive tracer into the modified amino acid. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated by reverse-phase HPLC, first in 0.1% trifluoroacetic acid with a gradient in acetonitrile and then in 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas-phase sequencing gave the modified peptide: Ser-Ala-X-Val-Pro-Val-Asp-Phe-Glu-Glu-Val-Val-Val-Ser-Ser-Asn-Ala-Asp-Gl u-Glu- Asp-Ile-Arg. The corresponding tryptic peptide that was isolated from unmodified enzyme yielded the same sequence except for (carboxymethyl)cysteine at position 3, suggesting that cysteine is the target of 3-bromo-2-ketoglutarate. Pig heart NAD+-dependent isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma) that can be separated by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The peptide modified by 3-bromo-2-ketoglutarate, which is in or near the substrate site, is derived only from the separated gamma subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.     

The mechanism of inhibition of Alcaligenes faecalis S-adenosylhomocysteine hydrolase by neplanocin A

Neplanocin A, a cyclopentenyl analog of adenosine, has been reported by S. Yaginuma, N. Muto, M. Tsujino, Y. Sudate, M. Hayashi, and M. Otari (1981) J. Antibiot. 34, 359-366 to exhibit antibacterial activity against Alcaligenes faecalis. Since neplanocin A (NpcA) is a known inhibitor of eukaryotic S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) (R. T. Borchardt, B. T. Keller, and U. Patel-Thombre (1984) J. Biol. Chem. 259, 4353-4358), the present study was undertaken to determine the effects of this carbocyclic nucleoside on AdoHcy hydrolase isolated from a prokaryotic source (A. faecalis). AdoHcy hydrolase was purified to homogeneity by affinity chromatography on an AdoHcy-agarose matrix from A. faecalis. Neplanocin A inactivated the purified AdoHcy hydrolase in a time- and concentration-dependent manner and the enzyme activity could not be recovered by dialysis. The inactivation of this bacterial enzyme by neplanocin A is accompanied by a reduction of three of the six enzyme-bound NAD+s to NADHs. These results suggest that the prokaryotic enzyme, like the eukaryotic AdoHcy hydrolase, is susceptible to inhibition by neplanocin A. The mechanism of inactivation in both cases appears to be a Kcat mechanism involving the reduction of the enzyme-bound NAD+ to NADH. The fact that total inhibition of the prokaryotic AdoHcy hydrolase by NpcA results in a reduction of only three of the six enzyme-bound NAD+s to NADHs suggests that the enzyme shows half-site reactivity (i.e., only three of the six subunits are catalytically active).     

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.     

Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv

An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Isolation and sequence of a peptide containing an essential lysine

Interaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with pyridoxal 5'-phosphate and sodium borohydride leads to inactivation and modification of two lysine residues per enzyme dimer that are thought to bind glucose 6-phosphate [Milhausen, M., & Levy, H.R. (1975) Eur. J. Biochem. 50, 453-461]. The amino acid sequence surrounding this lysine residue is reported. Following tryptic hydrolysis of the modified enzyme, two peptides, each containing one pyridoxyllysine residue, were purified to homogeneity and subjected to automated Edman degradation. The sequences revealed that one of these, a heptapeptide, was derived from the other, containing 11 amino acids. Supporting evidence for the role of the modified lysine is provided in the following paper [Haghighi, B., & Levy, H.R. (1982) Biochemistry (second paper of three in this issue)]. End-group analysis of the native enzyme revealed that valine is the N-terminal and glycine the C-terminal amino acid and provides support for the identity of the enzyme's two subunits.     

Enzyme-catalyzed DNA unwinding. The role of ATP in helicase III activity

The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G. T., Das, R. H., and Gefter, M. L. (1979) J. Biol. Chem. 254, 11997-12001). The free enzyme is able to bind to double- and single-stranded DNA. In the presence of ATP the enzyme can bind single- but not double-stranded DNA. The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA. It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA. These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis.     

Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP-sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility.