On the nucleotide binding domain of fructose-1,6-bisphosphatase.

Native chicken liver fructose-1,6-bisphosphatase (Fru-P₂ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P₂ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH₄ leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P₂ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P₂ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P₂ase, digestion of pyridoxal-P-Fru-P₂ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, M_r ~ 30,000, was found to contain all of the incorporated pyridoxal-P.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Rabbit liver fructose-1,6-bisphosphatase: location of an active site lysyl residue in the COOH-terminal fragment generated by a lysosomal proteinase

Digestion of native rabbit liver fructose-1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) with a membrane-bound proteinase from rat liver lysosomes yields a fragment of M_r = 9850. This peptide contains the COOH terminus of the Fru-P₂ase polypeptide chain and also the cyanogen bromide peptide (BrCN5) carrying the active site lysyl residue. The sequence of BrCN5 and its location with respect to the COOH terminus of the polypeptide chain have been determined. The active site lysyl residue is located at approximately residue ?54 from the COOH terminus. The bond hydrolyzed by the lysosomal proteinase is located between residues ?88 and ?89 from the COOH terminus.     

Pyridoxylation of essential lysine residues of mitochondrial adenosine triphosphatase

1. Soluble beef-heart mitochondrial ATPase (F₁) was incubated with [³H]pyridoxal 5′-phosphate and the Schiffbase complex formed was reduced with sodium borohydride. Spectral measurements indicate that lysine residues are modified and gel electrophoresis in the presence of detergent shows the tritium label to be associated with the two largest subunits, α and β. 2. In the absence of protecting ligands, the loss of ATP hydrolysis activity is linearly dependent on the level of pyridoxylation with complete inactivation correlating to 10 mol pyridoxamine phosphate incorporated per mol enzyme. Partial inactivation of F₁ with pyridoxal phosphate has no effect on either the K_m for ATP or the ability of bicarbonate to stimulate residual hydrolysis activity, suggesting a mixed population of fully active and fully inactive enzyme. 3. In the presence of excess magnesium, the addition of ADP or ATP, but not AMP, decreases the rate and extent of modification of F₁ by pyridoxal phosphate. The non-hydrolyzable ATP analog, 5′-adenylyl-β, γ-imidodiphosphate, is particularly effective in protecting F₁ against both modification and inactivation. Efrapeptin and P_i have no effect on the modification reaction. 4. Prior modification of F₁ with pyridoxal phosphate decreases the number of exchangeable nucleotide binding sites by one. However, pyridoxylation of F₁ is ineffective in displacing endogenous nucleotides bound at non-catalytic sites and does not affect the stoichiometry of P_i binding. 5. The ability of nucleotides to protect against modification and inactivation by pyridoxal phosphate and the loss of one exchangeable nucleotide site with the pyridoxylation of F₁ suggest the presence of a positively charged lysine residue at the catalytic site of an enzyme that binds two negatively charged substrates.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Immobilization of nucleoside diphosphatase at its allosteric site using immobilized derivatives of pyridoxal 5'-phosphate

3-O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH₄. 3-O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3-O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl₂ was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.     

磷酸吡哆醛修饰的蛇肌果糖1,6-二磷酸酯酶的时间分辨荧光分析

在别构抑制剂AMP或底物果糖1,6-二磷酸(FruP_2)存在下,磷酸吡哆醛(PLP)分别专一性地修饰在蛇肌果糖1,6-二磷酸酯酶(FruP_2ase,E.C.3.1.3.11.)的催化部位或别构部位。测得了修饰在催化部位或别构部位的PLP的荧光寿命及其连续分布。通过荧光寿命分布宽度的比较,认为该酶的活性部位柔性大于别构部位的柔性。     

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC₅₀) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.     

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.     

The bacterial wilt,uptake of phosphate,and phosphate ester levels in the resistant and susceptible alfalfa plants

7 days or 7 weeks old alfalfa plants (Medicago sativa L.), susceptible (S) and resistant (R) to bacterial wilt, were inoculated withCorynebacterium michiganense pv.insidiosum and on day 8 and 15 after inoculation the levels of acid-soluble phosphate esters (P-esters) were determinated by means of³²P labelling in the shoots or roots. The most significant changes were recorded in the roots of the older R plants grown in full Knop nutrient solutions on day 8 after inoculation. The marked reduction of inorganic phosphate (P₁) uptake by whole R plants is accompanied by a decrease in the levels of fructose-l, 6-bisphosphate (Fru-P₂), glucose-6-phosphate (Glc-6-P), fructose-6-phosphate (Fru-6-P), adenosine mono-, and diphosphate (AMP and ADP), phosphorylcholine (P-choline) and a proportional increase in the level of P₁. In the S plants, infection affected neither P₁ uptake nor P₁ proportions. In the plants grown after inoculation in diluted Knop’s solutions (0.147 mM KH₂PO₄), infection induced a reduction of the radial transport of P₁ to the segments of R roots whereas a reduction of the levels was only recorded in some P-esters [AMP, ADP, dihydroxyacetone phosphate (DHAP), and P-choline, but no decrease of Fru-P₂, Glc-6-P and Fru-6-P]. In the S plants, P₁ transport and the levels of P-esters were increased by the infection. P₁ transport exhibited considerable metabolic dependence (DNP, DCCD). Bacterial infection probably had no influence on the activity of the plasma membrane ATPases.     

Modification of the kinetics and regulatory properties of bovine hepatic fructose 1,6-diphosphatase with pyridoxal 5'-phosphate

Treatment of purified fructose 1,6-diphosphatase from bovine liver (which is maximally active at neutral pH) with pyridoxal 5'-phosphate produces changes in the spectral, catalytic, and allosteric properties of the enzyme. After modification the Michaelis constants for fructose-1,6-diphosphate and Mg²⁺ are increased, and inhibition by AMP is decreased. Substrate inhibition is decreased, but not abolished; the curve is merely shifted toward higher substrate concentration. Fructose-1, 6-diphosphate protects against the increases in the K_m for fructose-1, 6-diphosphate and the K_m for Mg²⁺, and against the changes in substrate inhibition, but not against the changes in AMP inhibition. AMP protects against the changes in AMP inhibition and the increase in the K_m for magnesium, but does not prevent the changes in substrate inhibition or the increase in the K_m for fructose-1, 6-diphosphate. The pH curves in the modified enzyme are altered at high, but not at low, substrate concentration.     

Location of the reactive sulfhydryl residues in the primary sequence of the β2 subunit of tryptophan synthase of Escherichiacoli

Two of the 5 sulfhydryl residues of the β₂ subunit of tryptophan synthase have previously been shown to react with N-ethylmaleimide and to have active site roles. We now show that the single sulfhydryl which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate is cysteine-170. The essential sulfhydryl which reacts with N-ethylmaleimide or with 2-nitro-5-thiocyanobenzoic acid after removal of pyridoxal phosphate is cysteine-230. The affinity reagent, bromoacetylpyridoxamine phosphate, reacts variably with cysteine-62 or with cysteine-230.     

Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5-p-fluorosulfonylbenzoyl adenosine.

The purine nucleotide derivative, 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSO₂B_ZAdo) functions as an affinity label for the allosteric sites of phosphofructokinase. The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites.     

Affinity labeling of the essential lysyl residue at the active site of enzymes by using nucleotide polyphosphate pyridoxal

We have discovered a new type of affinity labeling reagents for the nucleotide-binding site of protein by introducing an active site-directing moiety to pyridoxal 5-phosphate. Uridine diphosphopyridoxal almost completely inactivated glycogen synthase in a time-dependent manner (K _inact =25 µM;k ₀=0.22 min^–1). The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator Glc-6-P. The addition of cysteamine to the inactivated enzyme restored the original activity, whereas the treatment of the inactivated enzyme with NaBH₄ resulted in the fixation of the label to the enzyme protein. A peptide containing the label was isolated after proteolysis, and sequenced as E-V-A-K*-V-G-G-I-(Y). Adenosine polyphosphopyridoxal considerably inactivated lactate dehydrogenase in a time-dependent manner. The degree of inactivation was dependent on the number of phosphate groups; 64% (N=2), 51% (N=3), and 35% (N=4) at a reagent concentration of 1 mM for 30 min. The inactivation was protected by NADH, but not by pyruvate. Although the inactivation was not completed, the reagent was stoichiometrically incorporated into enzyme protein concomitantly with the inactivation. Affinity chromatographic analysis of the inactivated enzyme of Blue-Toyopearl revealed the presence of several protein species. The ratio of those species changed according to the stage of inactivation.     

Evidence for the conformation about the C(5′)-O(5′) bond of AMP complexed to AMP kinase: Substrate properties of a vinyl phosphonate analog of AMP

A vinyl phosphonate analog of adenosine 5′-phosphate (AMP) was synthesized in which the CH₂OP system of AMP is replaced by CHCHP. The V_max values of this analog relative to AMP were 0.7% with rabbit muscle AMP aminohydrolase, 13.4% with rabbit muscle AMP kinase, and 6.6% with pig muscle AMP kinase. The vinyl analog of ADP produced by the kinases was a substrate of rabbit muscle pyruvate kinase. These results, together with substrate specificity properties at the AMP sites of the enzymes indicate that the C(4′)-C(5′)-O(5′)-P system of AMP is of trans character during conversion of AMP to ADP by pig or rabbit AMP kinase.     

Nucleotide binding to glycogen phosphorylase b in the crystal.

The binding of the allosteric activator, AMP, and the inhibitor, ATP, to glycogen phosphorylase b has been studied in the crystal at 3 Å resolution. The nucleotides bind to two sites on the enzyme which are identified as site N, the allosteric effector site which is close to the subunit-subunit interface, and site I, a nucleoside inhibitor site which blocks the entrance to the active site crevasse. AMP when bound at the allosteric effector site makes several defined interactions with the enzyme in agreement with the results of solution studies. The contacts involve the N-10 position of the base, the 2′ hydroxyl of the ribose and the phosphate. IMP, analysed at 4 Å resolution, appears to bind in an identical conformation to AMP. At 3 Å resolution no well defined conformational changes are observed on binding AMP, although there are indications of a disturbance of the crystal lattice. It is concluded that the forces which stabilise the crystal lattice prevent the allosteric response of the enzyme in the crystal.     

Chemical modification of the bifunctional regulatory protein of maize leaf pyruvate,orthophosphate dikinase. Evidence for two distinct active sites

The active site(s) of the bifunctional regulatory protein of pyruvate,orthophosphate dikinase catalyze(s) the Pi-dependent activation (dephosphorylation) and ADP-dependent inactivation (phosphorylation) of maize leaf dikinase. The chemical modification studies of the regulatory protein active sites presented in this paper are interpreted as showing the two sites to be physically distinct. Pyridoxal 5'-phosphate and 2-nitro-5-thiocyanatobenzoate (NTCB) selectively inhibit the dikinase activating site, which is protected by the nonprotein substrate, Pi. Phenylglyoxal blocks both the activation and inactivation sites; the former is protected selectively by Pi and the latter by both the nonprotein substrate, ADP, and Pi. The Pi that protects the inactivation site is distinct from the activation substrate. Inhibition studies show Pi to be a parabolic competitive inhibitor of the ADP-dependent inactivation of dikinase, implying that besides substrate Pi, a second phosphate also binds to the regulatory protein. The above chemical modifications are not mutually exclusive; neither NTCB, 5,5'-dithiobis-(2-nitrobenzoate), nor pyridoxal 5'-phosphate blocks subsequent modification of the activation site by phenylglyoxal. Similarly, prior modification with NTCB does not affect modification by pyridoxal 5'-phosphate.     

Origin of tryptophan in fructose 1,6-bisphosphatase purified from livers of fed rabbits.

Fructose 1,6-bisphosphatase (Fru-P₂ase,EC 3.1.3.11) purified from livers of fed rabbits has been reported to contain tryptophan, which is not present in the enzyme purified from livers of fasted animals. We now show that the tryptophan arises from small amounts of active or inactive rabbit liver aldolase in the Fru-P₂ase preparations. Fru-P₂ase free of tryptophan may conveniently be prepared by raising the temperature of the heat step in the purification procedure to 67 °C.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.