Anomeric specificity of human liver and B-cell glucokinase: modulation by the glucokinase regulatory protein

The anomeric specificity of the wild-type recombinant forms of human liver and B-cell glucokinase was investigated using radioactive anomers of d-glucose as tracers. With d-glucose at anomeric equilibrium and at 30 degrees C, the maximal velocity, Hill number, and K(s) amounted, respectively, to 16 micromol min(-1) mg(-1), 1.8 and 6.9 mM in the case of liver glucokinase, and 7.3 micromol min(-1) mg(-1), 2.0 and 7.1 mM in the case of B-cell glucokinase. Whether at 20-22 or 30 degrees C, the maximal velocity, Hill number, and K(m) were significantly lower with alpha-d-glucose than with beta-d-glucose in both liver and B-cell glucokinase. As a result of these differences, the reaction velocity was higher with alpha-d-glucose at low hexose concentrations, while the opposite situation prevailed at high hexose concentrations. In the presence of 0.2 mM d-fructose 6-phosphate, the glucokinase regulatory protein caused a concentration-related inhibition of d-glucose phosphorylation, such an effect fading out at high concentrations of either d-glucose or glucokinase relative to that of its regulatory protein. The phosphorylation of alpha-d-glucose by liver glucokinase appeared more resistant than that of beta-d-glucose to the inhibitory action of d-fructose 6-phosphate, as mediated by the glucokinase regulatory protein. Such a phenomenon failed to achieve statistical significance in the case of the B-cell glucokinase. It is proposed that this information, especially the novel findings concerning the anomeric difference in both Hill number and sensitivity to the glucokinase regulatory protein, should be taken into account when considering the respective contributions of alpha- and beta-d-glucose to the overall phosphorylation of equilibrated d-glucose by glucokinase.     

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-d-glucopyranosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy-β-d-glucopyranose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from d-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.     

Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.     

alpha-Mannosidase-catalyzed synthesis of a (1-->2)-alpha-D-rhamnodisaccharide derivative

Enzymatic transglycosylation using p-nitrophenyl alpha-D-rhamnopyranoside as the glycosyl donor and 6equiv of ethyl 1-thio-alpha-D-rhamnopyranoside as the glycosyl acceptor yielded a D-rhamnooligosaccharide derivative. The reaction was catalyzed by jack bean alpha-mannosidase in a 1:1 (v/v) mixture of 0.1 M sodium citrate buffer (pH4.5)-MeCN at 25 degrees C. The enzyme exhibited high catalytic activity for the reaction, to afford in 32.1% isolated yield (based on donor substrate) ethyl alpha-D-rhamnopyranosyl-(1-->2)-1-thio-alpha-D-rhamnopyranoside, which is a derivative of the common oligosaccharide unit of the antigenic lipopolysaccharides from Pseudomonas.     

Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.     

Biochemistry of the initial steps of mycothiol biosynthesis

Mycothiol is the major thiol produced by mycobacteria and is required for growth of Mycobacterium tuberculosis. The final three steps in the biosynthesis of mycothiol have been fully elucidated but the initial steps have been unclear. A glycosyltransferase, MshA, is required for production of the mycothiol precursor, 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol, but its substrates and immediate products were unknown. In this study, we show that the N-acetylglucosamine donor is UDP-N-acetylglucosamine and that the N-acetylglucosamine acceptor is 1L-myo-inositol 1-phosphate. The reaction generates UDP and 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol 3-phosphate. Using cell-free extracts of M. smegmatis mc(2)155, little activity was obtained with myo-inositol, 1D-myo-inositol 1-phosphate, or myo-inositol 2-phosphate as the N-acetylglucosamine acceptor. A phosphatase, designated MshA2, is required to dephosphorylate 1-O-(2-acetamido-2-deoxy-alpha-glucopyranosyl)-D-myo-inositol 3-phosphate to produce 1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol. The latter is deacetylated, ligated with cysteine, and the cysteinyl amino group acetylated by acetyl-CoA to complete the mycothiol biosynthesis pathway. Uptake and concentration of myo-[14C]inositol is rapid in Mycobacterium smegmatis and leads to production of radiolabeled inositol 1-phosphate and mycothiol. This demonstrates the presence of a myo-inositol transporter and a kinase that generates 1L-myo-inositol 1-phosphate. The biochemical pathway of mycothiol biosynthesis is now fully elucidated.     

Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thaliana

The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1).     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Identification of a alpha-NeuAc-(2----3)-beta-D-galactopyranosyl N-acetyl-beta-D-galactosaminyltransferase in human kidney

Microsomal preparations from human kidney were found to contain enzymic activity capable to transfer N-acetylgalactosamine from UDP-N-acetylgalactosamine to native bovine fetuin. The acceptor structures on the fetuin molecules were identified as N- as well as O-linked glycans with a markedly higher incorporation into the N-linked carbohydrate chains. Analysis of the alkali-labile transferase products by thin-layer chromatography indicated that the enzyme is able to synthesize structures having mobilities identical with those found on glycophorin from Cad erythrocytes. Mild acid treatment and enzymic hydrolysis with N-acetylhexosaminidase from jack beans of the N-linked transferase products suggested that beta-D-GalpNAc-(1----4)-[alpha-NeuAc-(2----3)]-beta-D-Galp-(1----s tructures were formed by the enzymic reaction on both N- and O-linked acceptors. The enzyme might, therefore, be involved in the biosynthesis of Sda (and Cad) antigenic structures. By use of various oligosaccharides, glycopeptides, and glycolipids having well characterized carbohydrate sequences, the acceptor-substrate specificity of the N-acetylgalactosaminyltransferase was determined. The enzyme generally recognized alpha-NeuAc-(2----3)-beta-D-Gal groups as acceptors, but in a certain conformation. Thus, tri- and tetra-saccharide alditols, native human glycophorin A, and GM3 were not acceptor substrates although they carry the potential disaccharide acceptor unit. When these structures were presented as sialyl-(2----3)-lactose or as a tryptic peptide from glycophorin A, they were shown to be rather good acceptor substrates for the N-acetyl-beta-D-galactosaminyltransferase from human kidney.     

Mechanistic differences among retaining disaccharide phosphorylases: insights from kinetic analysis of active site mutants of sucrose phosphorylase and alpha,alpha-trehalose phosphorylase

Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.     

A new erythrose 4-phosphate dehydrogenase coupled assay for transketolase

The standard assay for transketolase (E.C 2.2.1.1) has depended upon the use of d-xylulose 5-phosphate as the ketose donor substrate since the production of d-glyceraldehyde 3-phosphate can be readily coupled to a reaction that consumes NADH allowing the reaction to be followed spectrophotometrically. Unfortunately, commercial supplies of d-xylulose 5-phosphate recently became unavailable. In this article we describe the coupling of a transketolase reaction (using Leishmania mexicana transketolase) that converts d-fructose 6-phosphate to d-erythrose 4-phosphate. d-Erythrose 4-phosphate can then be converted to 4-phosphate d-erythronate using erythrose-4-phosphate dehydrogenase (E.C 1.2.1.72), a reaction that reduces NAD⁺ to NADH and can be easily followed spectrophotometrically. d-Ribose 5-phosphate and d-glyceraldehyde 3-phosphate can both be used as ketol acceptor substrates in the reaction although d-ribose 5-phosphate is also a substrate for the coupling enzyme.     

A bisubstrate analog inhibitor for alpha(1----2)-fucosyltransferase

Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.     

A lack of reactivity of d-arabinose 5-phosphate with enzymes of the pentose phosphate pathway

The reported conversion of d-arabinose 5-phosphate to d-ribose 5-phosphate and other intermediates of the pentose phosphate pathway was investigated. Two new solvent systems to separate the two aldopentose phosphates on paper and a method using chromatography on a column of dihydroxyboryl-cellulose were developed. No evidence for their interconversion could be obtained. d-Arabinose 5-phosphate did not serve as an acceptor for transketolase from bakers' yeast, Candida utilis, or rat liver but behaved as an inhibitor. d-Glucose 6-phosphate acted both as an acceptor and as an inhibitor of the reaction with d-ribose 5-phosphate as acceptor. d-Arabinose 5-phosphate was not converted into ribose 5-phosphate, ketopentose phosphate, triose phosphate, or a heptulose phosphate by rat muscle or rat liver enzymes. Hydroxypyruvate is suggested not to be a substrate for rat liver transketolase.     

Escherichia coli phnN,encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.     

Regiospecific glycosidase-assisted synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 3'-sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta1-3GlcNAc).

The all-transglycolytic synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 3'-sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta1-3GlcNAc) was performed. The disaccharide lacto-N-biose I was obtained by use of p-nitrophenyl beta-D-galactopyranoside as the donor, 2-acetamido-2-deoxy-D-glucopyranose as the acceptor and Xanthomonas manihotis beta-D-galactosidase as the catalyst. The reaction was shown to be regiospecific, with a high molar yield (about 55%) with respect to the donor. Lacto-N-biose I obtained by this method was used as the acceptor for a subsequent enzymatic reaction catalyzed by Trypanosoma cruzi trans-sialidase in which 2'-(4-methylumbellyferyl)-alpha-D-N-acetylneuraminic was used as the donor of the N-acetylneuraminil moiety. The reaction generated the product, 3'-sialyl-lacto-N-biose I, regiospecifically and with a molar yield of about 35%.     

The trans-sialidase from Trypanosoma cruzi efficiently transfers alpha-(2-->3)-linked N-glycolylneuraminic acid to terminal beta-galactosyl units

The trans-sialidase from Trypanosoma cruzi (TcTS), the agent of Chagas' disease, is a unique enzyme involved in mammalian host-cell invasion. Since T. cruzi is unable to synthesize sialic acids de novo, TcTS catalyzes the transfer of alpha-(2-->3)-sialyl residues from the glycoconjugates of the host to terminal beta-galactopyranosyl units present on the surface of the parasite. TcTS also plays a key role in the immunomodulation of the infected host. Chronic Chagas' disease patients elicit TcTS-neutralizing antibodies that are able to inhibit the enzyme. N-Glycolylneuraminic acid has been detected in T. cruzi, and the trans-sialidase was pointed out as the enzyme involved in its incorporation from host glycoconjugates. However, N-glycolylneuraminic acid alpha-(2-->3)-linked-containing oligosaccharides have not been analyzed as donors in the T. cruzi trans-sialidase reaction. In this paper we studied the ability of TcTS to transfer N-glycolylneuraminic acid from Neu5Gc(alpha2-->3)Gal(beta1-->4)GlcbetaOCH(2)CH(2)N(3) (1) and Neu5Gc(alpha2-->3)Gal(beta1-->3)GlcNAcbetaOCH(2)CH(2)N(3) (2) to lactitol, N-acetyllactosamine and lactose as acceptor substrates. Transfer from 1 was more efficient (50-65%) than from 2 (20-30%) for the three acceptors. The reactions were inhibited when the enzyme was preincubated with a neutralizing antibody. K(m) values were calculated for 1 and 2 and compared with 3'-sialyllactose using lactitol as acceptor substrate. Analysis was performed by high-performance anion-exchange (HPAEC) chromatography. A competitive transfer reaction of compound 1 in the presence of 3'-sialyllactose and N-acetyllactosamine showed a better transfer of Neu5Gc than of Neu5Ac.     

Effect of nucleotide substitution on the peptidyltransferase activity of 2'(3')-O-(aminoacyl) oligonucleotides

Seven 2'(3')-O-(aminoacyl) trinucleotides with structures derived from the 3'-terminal C-C-A sequence of aa-tRNA via nucleotide substitutions were investigated as acceptor substrates in the peptidyltransferase reaction and as inhibitors of substrate binding to the peptidyltransferase A site. It was found that all tested compounds were active in both systems, although substitution in the first and second nucleotide position results in some decrease of acceptor activity. Remarkably, replacement of natural cytidylic acid residues in C-C-A-Phe with guanylic acid moieties resulted only in a small decrease of acceptor or binding activity. The results indicate that the acceptor sequence of aa-tRNA is not probably engaged in base pairing with a sequence of 23S RNA during its interaction with the peptidyltransferase A site.     

Enzymatic alpha-glucosaminylation of maltooligosaccharides catalyzed by phosphorylase

This paper describes phosphorylase-catalyzed enzymatic alpha-glucosaminylation for the direct incorporation of a 2-amino-2-deoxy-alpha-d-glucopyranose unit into maltooligosaccharides. When the reaction of 2-amino-2-deoxy-alpha-d-glucopyranosyl 1-phosphate as the glycosyl donor with maltotetraose as a glycosyl acceptor was performed in the presence of phosphorylase, glucosaminylated oligosaccharides were produced, which were characterized by MALDI-TOF MS measurement after N-acetylation of the crude products. The N-acetylated derivative of the main product in this system was isolated by using HPLC, and its structure was confirmed by MS and (1)H NMR spectra. Furthermore, glucoamylase-catalyzed reaction of the isolated compound provided support that the alpha-glucosamine unit is positioned at the non-reducing end of the oligosaccharide.     

Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity

A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The Km values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mm for fructose 6-phosphate and 1.2 mm for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMP-dependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.