Modification of the ATP inhibitory site of the Ascaris suum phosphofructokinase results in the stabilization of an inactive T state.

Treatment of the Ascaris suum phosphofructokinase (PFK) with 2',3'-dialdehyde ATP (oATP) results in an enzyme form that is inactive. The conformational integrity of the active site, however, is preserved, suggesting that oATP modification locks the PFK into an inactive T state that cannot be activated. A rapid, irreversible first-order inactivation of the PFK is observed in the presence of oATP. The rate of inactivation is saturable and gives a KoATP of 1.07 +/- 0.27 mM. Complete protection against inactivation is afforded by high concentrations of ATP, and the dependence of the inactivation rate on the concentration of ATP gives a Ki of 326 +/- 26 microM for ATP which is 22-fold higher than the Km for ATP at the catalytic site but close to the binding constant for ATP to the inhibitory site. Fructose 6-phosphate, fructose 2,6-bisphosphate, and AMP provide only partial protection against modification. The pH dependence of the inactivation rate gives a pKa of 8.4 +/- 0.1. Approximately 2 mol of [3H]oATP is incorporated into a subunit of PFK concomitant with 90% loss of activity, and ATP prevents the derivatization of 1 mol/subunit. The oATP-modified enzyme is not activated by AMP or fructose 2,6-bisphosphate. oATP has no effect on the activity of a desensitized form of PFK in which the ATP inhibitory site is modified with diethyl pyrocarbonate but with the active site intact [Rao, G.S.J., Wariso, B.A., Cook, P.F., Hofer, H.W., & Harris, B.G. (1987) J. Biol. Chem. 262, 14068-14073].(ABSTRACT TRUNCATED AT 250 WORDS)     

Halo enol lactones: studies on the mechanism of inactivation of alpha-chymotrypsin

In a previous investigation [Daniels, S. B., Cooney, E., Sofia, M. J., Chakravarty, P. K., & Katzenellenbogen, J. A. (1983) J. Biol. Chem. 258, 15046-15053], we demonstrated that alpha-aryl-substituted five- and six-membered ring halo enol lactones were effective inhibitors of chymotrypsin, and we proposed that they reacted by an enzyme-activated mechanism: acyl transfer to the active site serine generates a halomethyl ketone that remains tethered in the catalytic site until it alkylates an accessible nucleophilic residue. In this study, we have investigated in greater detail the process of chymotrypsin inactivation by an alpha-naphthyl-substituted five- and six-membered bromo enol lactone. Inactivation by both compounds appears to be active site directed, since the time-dependent inactivation is retarded by competing substrate. The possible involvement of a paracatalytic mechanism for inactivation (generation of a free, rather than active site bound, inactivating species) was investigated by comparing the inactivation efficiencies of the lactones with that of the bromomethyl keto acid hydrolysis products. The bromomethyl ketone derived from the five-membered lactone is ineffective, whereas that derived from the six-membered lactone is highly efficient. However, the possible involvement of the free keto acid in chymotrypsin inactivation by the six-membered lactone is ruled out by experiments involving selective scavenging. The long-term inactivation of chymotrypsin requires the presence of the bromine substituent and appears to involve an alkylation rather than an acylation reaction (hydrazine resistant). Furthermore, a 1:1 lactone:enzyme stoichiometry is demonstrated with the 14C-labeled six-membered lactone. These results are consistent with the mechanism-based inactivation process previously presented.     

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.     

Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.     

Adenine nucleoside dialdehydes: potent inhibitors of bovine liver S-adenosylhomocysteine hydrolase

Various ribonucleoside 2',3'-dialdehydes, including adenosine dialdehyde, S-adenosylhomocysteine (AdoHcy) dialdehyde, and 5-(methylthio)-5'-deoxyadenosine (MTA) dialdehyde, were shown to be potent inhibitors of bovine liver AdoHcy hydrolase (EC 3.3.1.1). These ribonucleoside 2',3'-dialdehydes produce both time-dependent and concentration-dependent inactivation of the AdoHcy hydrolase. The inactivation appears to be irreversible since the enzyme activity cannot be recovered after prolonged dialysis against phosphate buffer. However, a substantial percentage of the enzyme activity could be recovered when the inactivated enzyme was dialyzed against a nitrogen buffer [e.g., tris(hydroxymethyl)aminomethane (Tris)]. This reversal of inhibition could be prevented, however, by pretreatment of the ligand-enzyme complex with sodium borohydride prior to dialysis in Tris buffer. Inclusion of substrates (e.g., adenosine or AdoHcy) afforded protection of the enzyme from the inactivation induced by the ribonucleoside 2',3'-dialdehydes. These data suggest that the bond formed between the enzyme and the inhibitor is probably a Schiff base linkage between the aldehydic functionality of the inhibitor and a protein lysinyl residue in or around the adenosine-AdoHcy binding site. When [2,8-3H]adenosine dialdehyde was used, a stoichiometry of 1.73 nmol of inhibitor bound per nmol of AdoHcy hydrolase was determined. Analysis of the kinetics of enzyme inactivation using the Ackermann-Potter approach indicates that adenosine dialdehyde is a tight-binding inhibitor, exhibiting a stoichiometry of one to two molecules of inhibitor bound to one molecule (tetramer) of enzyme and a Ki = 2.39 nM.     

Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding.

The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied. The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS. The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect. Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition. However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS. These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A. (1990) J. Biochem. 108, 572-578), might be involved in the active site or be located in the vicinity of the active site. The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.     

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.     

Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine

Non-acetylated glucosamine is an unusual structural feature shared by all glycosyl phosphatidylinositol (GPI) lipids, including a variety of membrane anchors, the leishmanial lipophosphoglycan, and a mediator of insulin action. We proposed previously a pathway for biosynthesis of glycolipid A, the precursor of the GPI membrane anchor of the trypanosome variant surface glycoprotein (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800). In this paper we characterize in more detail the initial steps of GPI assembly. The first and committed step in the pathway is the transfer of GlcNAc, from UDP-GlcNAc, to endogenous phosphatidylinositol to form N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). The GlcNAc-PI is then efficiently deacetylated to form glucosaminyl phosphatidylinositol (GlcN-PI), the substrate for subsequent reactions en route to glycolipid A.     

Significance of phosphorylation of phosphofructokinase

In order to understand the effect of phosphorylation on phosphofructokinase, the allosteric kinetic behavior, ligand binding at various pHs, and pH-dependent cold inactivation of phosphofructokinase phosphorylated to different extents were studied. A subtilisin-digested phosphofructokinase from which a COOH-terminal peptide containing a phosphorylation site has been cleaved (Riquelme, P. T., and Kemp, R. G. (1980) J. Biol. Chem. 255, 4367-4371) was also included in these studies in order to investigate the possible role of this region of the molecule. Allosteric kinetics and direct binding experiments have shown that increasing phosphorylation of phosphofructokinase results in increased sensitivity to ATP inhibition and stronger binding of ATP to the inhibitory site of the enzyme. Ths subtilisin-cleaved phosphofructokinase is the least sensitive to the inhibition and shows the weakest binding of ATP. The opposite effect is observed with the binding isotherms of fructose-6-P. There is no difference in the binding of fructose-2,6-P2 among these enzymes. Binding of ATP to the inhibitory site of these enzymes as determined by fluorescence quenching (Pettigrew, D. W., and Frieden, C. (1979) J. Biol. Chem. 254, 1887-1895) is affected by pH; the binding is greatly enhanced at lower pH. Moreover, there is little difference in the binding among the modified enzymes at pH 8, but at lower pHs the binding to the phosphorylated enzyme is much more enhanced than the dephosphoenzyme. A pH-dependent cold inactivation study has shown that the phosphorylation of the enzyme causes an increase in the pK value for the inactivation, and the extent of the pK shift depends upon the degree of phosphorylation. Based on these results, a model originally proposed by Frieden et al. (Frieden, C., Gilbert, H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647) can be applied to explain a possible role for the phosphorylation and the peptide portion of phosphofructokinase in its complex allosteric kinetic behavior.     

Acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for an active site histidine residue

The lysosomal membrane enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase catalyzes the transfer of the acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction appears to be a transmembrane process: the enzyme is acetylated on the outside of the lysosome, and the acetyl group is transferred across the membrane to the inside of the lysosome where it is used to acetylate glucosamine. To determine the reactive site residues involved in the acetylation reaction, lysosomal membranes were treated with various amino acid modification reagents and assayed for enzyme activity. Although four thiol modification reagents were examined, only one, p-chloromercuribenzoate inactivated the N-acetyltransferase. Thiol modification by p-chloromercuribenzoate did not appear to occur at the active site since inactivation was still observed in the presence of the substrate acetyl-CoA. N-Acetyltransferase could be inactivated by N-bromosuccinimide, even after pretreatment with reagents specific for tyrosine and tryptophan, suggesting that the modified residue is a histidine. Diethyl pyrocarbonate, another histidine modification reagent, could also inactivate the enzyme; this inactivation could be reversed by incubation with hydroxylamine. N-Bromosuccinimide and diethyl pyrocarbonate modifications appear to be at the active site of the enzyme since co-incubation with acetyl-CoA protects the N-acetyltransferase from inactivation. This protection is lost if glucosamine is also present. Pre-acetylated lysosomal membranes are also able to provide protection from N-bromosuccinimide inactivation, providing further evidence for a histidine moiety at the active site and for the existence of an acetyl-enzyme intermediate.     

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.     

The adenine nucleotide-binding site on yeast 3-phosphoglycerate kinase. Affinity labeling of Lys-131 by pyridoxal 5'-diphospho-5'-adenosine

The adenine nucleotide analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) is a potent and highly specific inactivator of yeast 3-phosphoglycerate kinase. Supportive evidence includes the finding that 1) during a 10-min incubation, half-maximal inactivation is given by 10 microM PLP-AMP, 2) covalent incorporation of 1.2 mol of PLP-AMP/mol of enzyme is sufficient to give complete inactivation, and 3) MgATP gives near complete protection against modification and inactivation by PLP-AMP. Following reaction with PLP-AMP and reduction with NaBH4 to form a stable adduct, the enzyme was digested with endoproteinase Lys-C and peptides were separated by reversed-phase high-performance liquid chromatography. The single major labeled peptide was purified and sequenced, and the modified residue was identified as Lys-131. The crystal structure of enzyme in the open conformation shows Lys-131 to reside within a loop of flexible random coil positioned at the outer edge of the central binding cleft, approximately 2 nm from the surface of the cleft that comprises part of the MgATP-binding site (Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., and Kingsman, S. M. (1982) EMBO J. 1, 1635-1640). We conclude that the structural element containing Lys-131 undergoes substantial movement during the ligand-induced conformational change known to occur during formation of the ternary complex, resulting in the positioning of a basic residue near a negatively charged substrate. Since similar affinity-labeling results have been presented for hexokinase (Tamura, J. K., LaDine, J. R., and Cross, R. L. (1988) J. Biol. Chem. 263, 7907-7912), we further suggest that movement of positive charge into the central cleft may be a common step in the tight binding of nucleotides by bilobal kinases.     

Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis.

Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and P(i), the fifth step in de novo purine biosynthesis. The ATP binding domain of the E. coli enzyme has been investigated using the affinity label [(14)C]-p-fluorosulfonylbenzoyl adenosine (FSBA). This compound results in time-dependent inactivation of the enzyme which is accelerated by the presence of FGAM, and gives a K(i) = 25 microM and a k(inact) = 5.6 x 10(-)(2) min(-)(1). The inactivation is inhibited by ADP and is stoichiometric with respect to AIR synthetase. After trypsin digestion of the labeled enzyme, a single labeled peptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA. Site-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gln, a Leu, and an Arg and the kinetic parameters of the mutant proteins were measured. All three mutants gave k(cat)s similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild-type enzyme. Efforts to inactivate the chicken liver trifunctional AIR synthetase with FSBA were unsuccessful, despite the presence of a Lys27 equivalent. The role of Lys27 in ATP binding appears to be associated with the methylene linker rather than its epsilon-amino group. The specific labeling of the active site by FSBA has helped to define the active site in the recently determined structure of AIR synthetase [Li, C., Kappock, T. J., Stubbe, J., Weaver, T. M., and Ealick, S. E. (1999) Structure (in press)], and suggests additional flexibility in the ATP binding region.     

S-adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine

Homogeneous S-adenosylhomocysteinase (AdoHcyase) from rat liver is a tetrameric enzyme that contains four molecules of tightly bound NAD per mole of enzyme. We report here that incubation of the rat liver enzyme with ATP, Mg2+, and KCl leads to conversion of the active enzyme to an inactive form with release of all enzyme-bound NAD which can be recovered quantitatively by gel filtration. At various concentrations of ATP, the release of NAD corresponds closely with the degree of inactivation, suggesting that the four subunits are equivalent. Hydrolysis of ATP is not required for the inactivation process since nonhydrolyzable ATP analogues can replace ATP in the inactivation process. The ATP-dependent inactivation is fully reversible upon incubation of the inactivated enzyme with NAD. The ATP-dependent inactivation of the enzyme appears to be analogues to the cAMP-dependent inactivation of the enzyme from Dictyostelium discoideum described earlier by Hohman et al. (1985) [Hohman, R. J., Guitton, M. C., & Veron, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4578-4581; Hohman, R. J., Veron, M., & Guitton, M. C. (1985) Curr. Top. Cell. Regul. 26, 233-245] but differs from the irreversible inactivation studied earlier by Abeles et al. (1982) [Abeles, R. H., Fish, S., & Lapinskas, B. (1982) Biochemistry 21, 5557-5562]. These authors have ascribed the time-dependent inactivation that results from incubation of the enzyme with 2'-deoxyadenosine at the C-3' and concluded that AdoHcyase "probably consists of two nonequivalent pairs of subunits".(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

Affinity labeling of adenylate kinase with adenosine diphosphopyridoxal. Presence of Lys21 in the ATP-binding site

Adenosine diphosphopyridoxal, the affinity labeling reagent specific for a lysyl residue in the nucleotide-binding site of several enzymes (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964; Tamura, J. K., Rakov, R. D., and Cross R. L. (1986) J. Biol. Chem. 261, 4126-4133) was applied to adenylate kinase from rabbit muscle. Incubation of the enzyme with a low concentration of the reagent at 25 degrees C for 20 min followed by reduction by sodium borohydride resulted in rapid inactivation of the enzyme. Extrapolation to 100% loss of enzyme activity gave a value of 1.0 mol of the reagent per mol of enzyme. ADP, ATP, and MgATP almost completely protected the enzyme from inactivation, whereas AMP offered little retardation of the inactivation. Dilution of the inactivated enzyme which had not been treated with the reducing reagent led to restoration of enzyme activity. This reactivation was accelerated by ATP but not by AMP. Structural study of the labeled peptide showed that Lys21 is exclusively labeled by adenosine diphosphopyridoxal. These results suggest that the epsilon-amino group of Lys21 is located in the ATP-binding site of the enzyme, more specifically at or close to the subsite for the gamma-phosphate of the nucleotide.     

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin

We have shown previously (Brooker, R.J., and Slayman, C.W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226) that the plasma membrane [H+]-ATPase of Neurospora crassa is inhibited by N-ethylmaleimide (NEM), which reacts at an essential nucleotide-protectable site on the Mr = 104,000 polypeptide. The present study demonstrates that Mg2+ has a biphasic effect on NEM inhibition. At low concentrations (0.01-0.1 mM, Mg2+ decreases the sensitivity of the enzyme to NEM, while at high concentrations (greater than 1 mM), it enhances sensitivity. These effects are seen in the presence or absence of nucleotides (ATP, ADP). Mg2+ also acts in a concentration-dependent way to influence the degradation of the ATPase by trypsin. Low concentrations of Mg2+ have little or no effect on tryptic inactivation of ATPase activity or on the disappearance of the Mr = 104,000 polypeptide and the stepwise appearance of Mr = 100,000 and 91,000 tryptic fragments. High concentrations of Mg2+ decrease the rate of inactivation, and a new fragment of Mr = 98,000 is seen. Taken together, the NEM and trypsin results indicate that the Neurospora [H+]-ATPase possesses high and low affinity Mg2+ binding sites which affect the conformation of the enzyme. The divalent cation specificity of the sites has also been investigated. Co2+, Mn2+, and (to a lesser extent) Ni2+ mimic the behavior of Mg2+, but Ca2+ has a different effect, at least at the high affinity site. It appears to bind to that site, based on its ability to inhibit ATP hydrolysis (in the presence of Mg2+), but does not offer protection against NEM inhibition. The results suggest a way in which Ca2+ may serve as a physiological regulator of the ATPase.     

Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.     

Complete structure of the glycan of lipopeptidophosphoglycan from Trypanosoma cruzi Epimastigotes.

The lipopeptidophosphoglycan is the major cell surface glycoconjugate of the epimastigote forms of the parasitic protozoan Trypanosoma cruzi. A detailed partial structure for this molecule has been reported (Previato, J. O., Gorin, P. A. J., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M., and Mendonca-Previato, L. (1990) J. Biol. Chem. 265, 2518-2526). In this study, we complete the primary structure assignments and describe the microheterogeneity found in the lipopeptidophosphoglycan glycan, using a combination of 1H and 31P NMR, fast atom bombardment mass spectrometry, methylation linkage analysis, and exoglycosidase sequencing. The lipopeptidophosphoglycan is a glycosylated inositol-phosphoceramide with striking homology to glycosylphosphatidylinositol membrane anchors found attached to a wide variety of plasma membrane proteins throughout the eukaryotes.     

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Active enzyme ultracentrifugation studies.

Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an S20,W value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme (S20,W = 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan, N. O. (1970), J. Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 651-664), appears, therefore, to be an inactive species. The physiological implications of the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.