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J F Robyt

Publications and source records attributed to J F Robyt.

At least 37 records · Page 2Linked to original sources
PubMed1990

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen.

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.

Glucosyltransferases↗
PubMed1990

Acceptor reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase.

The acceptor products of maltose with Leuconostoc mesenteroides B-512FM dextransucrase are panose (6(2)-alpha-D-glucopyranosyl maltose) and a homologous series of 6(2)-isomaltodextrinosyl maltoses. The structures of the acceptor products of dextransucrase with other maltodextrins, maltotriose to maltooctaose (G3-G8), were determined by using the known specificities of alpha-glucosidase and porcine pancreatic alpha-amylase, and by methylation analysis. It has been found that dextransucrase transfers a D-glucopyranosyl residue to C-6 of either the nonreducing end or the reducing end residues of the maltodextrins, G3-G8, forming an alpha(1----6) linkage. When a D-glucose was transferred to the nonreducing residue, the first product was also an acceptor to give the second product, which served as an acceptor to give the third product, etc. to give a homologous series. When D-glucose was transferred to the reducing residue, the first product did not readily serve as an acceptor to give products or it served only as a very poor acceptor to give a small amount of the next homologue. The effectiveness of maltodextrins as acceptors decreased as the size of the maltodextrin chain increased. Maltotriose was 40% as effective as maltose and maltooctaose was only 6% as effective.

Autoradiography↗
PubMed1990

A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase.

Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine. The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain. B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity. The procedure has been used to produce purified enzyme for sequencing. The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.

Chromatography, Gel↗
PubMed1989

Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance.

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.

Animals↗
PubMed1989

Synthesis of 4,6-dideoxysucrose, and inhibition studies of Leuconostoc and Streptococcus D-glucansucrases with deoxy and chloro derivatives of sucrose modified at carbon atoms 3, 4, and 6.

Starting from sucrose, 2,3,1',3',4',6'-hexa-O-benzoyl-6-deoxy-6-iodosucrose (1) was synthesized. Reaction of 1 with sulfuryl chloride in pyridine gave 2,3,1',3',4',6'-hexa-O-benzoyl-4-chloro-4,6-dideoxy-6-iodogalactosucr ose (2). Compound 2 was treated with tributyltin hydride in toluene in the presence of a radical initiator, alpha, alpha-azobis(isobutanonitrile) (AIBN), to remove iodine and chlorine groups and give hexa-O-benzoyl-4,6-dideoxysucrose. Benzoyl groups were removed by sodium methoxide in methanol to give 4,6-dideoxysucrose. Sucrose was modified at carbon atom 3, carbon atom 4, or carbon atoms 4 and 6, and these analogs were tested as inhibitors of the D-glucansucrases (D-glucosyltransferases) of Streptococcus mutans 6715 and Leuconostoc mesenteroides B-512F. Sucrose analogs used in this study are 4-deoxysucrose and 4-chloro-4-deoxygalactosucrose with S. mutans 6715 D-glucansucrases (GTF-S and GTF-I), and 3-deoxysucrose, 4-deoxysucrose, 4-chloro-4-deoxygalactosucrose, 6-deoxysucrose, and 4,6-dideoxysucrose with L. mesenteroides B-512F D-glucansucrase. The data indicate that 3-deoxysucrose, 4-deoxysucrose, and 4-chloro-4-deoxygalactosucrose are weak noncompetitive inhibitors for B-512F dextransucrase, with Ki values of 530, 201, and 202mM respectively. For the same enzyme, 6-deoxysucrose was a strong competitive inhibitor, with Ki of 1.60mM, and 4,6-dideoxysucrose was a good competitive inhibitor, with Ki of 20.3mM. 4-Deoxysucrose was a weak noncompetitive inhibitor for both GTF-I and GTF-S, with Ki values of 672 and 608mM, respectively. 4-Chloro-4-deoxygalactosucrose was also a weak noncompetitive inhibitor for GTF-I and GTF-S with Ki values of 391 and 308mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucosyltransferases↗
PubMed1988

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation.

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.

Carbohydrates↗
PubMed1988

Neisseria perflava amylosucrase: characterization of its product polysaccharide and a study of its inhibition by sucrose derivatives.

Neisseria perflava amylosucrase forms from sucrose a polysaccharide very similar to glycogen, except that a larger proportion of its D-glucosyl residues are in short branches. Iodine staining of samples taken during polysaccharide formation indicate that the initial product is less branched than that formed at longer times. This glycogen-like polysaccharide has an estimated molecular mass range of 1 MD to 20 MD. Sucrose derivatives modified at C-3 (3-deoxysucrose and alpha-D-allopyranosyl beta-D-fructofuranoside), C-6 (6-deoxysucrose and 6-deoxy-6-fluorosucrose), and both C-4 and C-6 (4,6-dideoxysucrose) were tested as inhibitors of amylosucrase. Derivatives modified at C-6 were potent competitive inhibitors, with Ki values of 6.2 +/- 0.3 mM (6-deoxysucrose) and 0.50 +/- 0.06 mM (6-deoxy-6-fluorosucrose). The KM value of sucrose is 26.5 +/- 4.6 mM. Sucrose derivatives modified at C-3 were not significantly inhibitory over the concentration range tested. 4,6-Dideoxysucrose gave an unusual, non-competitive inhibition, in that, increasing its concentration did not produce a commensurate increase in the level of inhibition, which instead appeared to approach a limit. None of these sucrose derivatives was a substrate for amylosucrase, nor were they glycosyl donors to maltotriose.

Glucosyltransferases↗
PubMed1986

Inhibition- and acceptor-reaction studies of Streptococcus mutans 6715 glucosyltransferases with 3-deoxysucrose, 3-deoxy-3-fluorosucrose, and alpha-D-allopyranosyl beta-D-fructofuranoside.

Three new sucrose analogs modified at C-3 have been studied as inhibitors and substrates for the glucosyltransferases (glucansucrases) of Streptococcus mutans 6715. Although none of the analogs were found to be substrates for polymer synthesis with either the soluble-polysaccharide producing enzyme, GTF-S, or the insoluble-polysaccharide producing enzyme, GTF-I, 3-deoxysucrose and 3-deoxy-3-fluorosucrose were able to donate glycosyl residues for acceptor reactions with both enzymes. Modification at C-3 considerably decreased the binding at the active site of both enzymes, since all of the analogs had inhibition constants at least one order of magnitude greater than the Km value for sucrose.

Chromatography, Thin Layer↗
PubMed1986

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention.

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.

Chymotrypsin↗
PubMed1986

Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris).

Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.

Binding Sites↗
PubMed1986

Functional molecular size and structure of dextransucrase by radiation inactivation and gel electrophoresis.

Robyt et al. have proposed a mechanism for dextransucrase in which dextran is synthesized by the cooperative action of two equivalent nucleophiles (Robyt, J.F., Kimble, B.K. and Walseth, T.F. (1974) Arch. Biochem. Biophys. 165, 634-640). To distinguish between the possibilities that the enzyme is a monomer bearing both nucleophiles, or a dimer with each subunit bearing one nucleophile, the molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis and by radiation inactivation. Two major forms of dextransucrase from Leuconostoc mesenteroides NRRL B-512F were found on SDS-polyacrylamide gel electrophoresis, with Mr 177 000 and 158 000, and sometimes a minor form with Mr 168 000. No form of dextransucrase smaller than Mr 158 000 was found, either in the presence or absence of dextran T10, although levansucrase was detected at Mr 92 000 and 116 000. On irradiation with 60Co, dextransucrase behaved as a single species with a maximum size of Mr 201 000. Because Mr 201 000 is much smaller than the minimum dimer size of Mr 316 000 (= 2 X 158 000), it is concluded that both nucleophiles are probably located on the same peptide, rather than one on each subunit of a dimer, and that peptide association is probably not required for dextran synthesis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.

Chromatography, Affinity↗
PubMed1986

Activation and inhibition of dextransucrase by calcium.

Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.

Calcium↗
PubMed1985

Porcine-pancreatic alpha amylase hydrolysis of substrates containing 6-deoxy-D-glucose and 6-deoxy-6-fluoro-D-glucose and the specificity of subsite binding.

Hydrolysis of 6-deoxyamylose and mono-6-deoxy-6-fluorocyclomaltoheptaose by porcine-pancreatic alpha amylase produces low-molecular-weight modified products, which have been analyzed by chemical and chromatographic techniques. Results for both substrates show that modified D-glucose and two isomers of modified maltoses are produced in the enzyme reaction. In addition, the formation of maltoses modified in the nonreducing residue is more favored than the formation of maltoses modified in the reducing residue. These results indicate that productive binding of 6-fluoro- and 6-deoxy-D-glucose residues is permitted at subsites 1 through 4 of the amylase-active site but that binding of these modified residues may be less favorable at subsite 3, the subsite at which catalytic attack occurs.

Amylose↗
PubMed1985

The effect of substrate modification on binding of porcine pancreatic alpha amylase: hydrolysis of modified amylose containing D-allose residues.

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.

Amylose↗
PubMed1985

Inhibition of Streptococcus mutans 6715 glucosyltransferases by sucrose analogs modified at positions 6 and 6'.

Sucrose derivatives modified at position 6 (6-deoxysucrose, 6-thiosucrose, 6,6'-dithiodisucrose, and 6,6'-dideoxy-6,6'-difluorosucrose) were tested as inhibitors of the two Streptococcus mutans 6715 glucosyltransferases. 6-Deoxysucrose was the best inhibitor studied, competitively inhibiting the soluble-D-glucan forming enzyme (GTF-S) and the insoluble-D-glucan forming enzyme (GTF-I) with Ki values one order of magnitude lower than the sucrose Km values. 6-Thiosucrose was also a competitive inhibitor for both enzymes. 6,6'-Dithiodisucrose and 6,6'-dideoxy-6,6'-difluorosucrose only inhibited GTF-I; 6,6'-dithiodisucrose gave mixed inhibition and 6,6'-dideoxy-6,6'-difluorosucrose gave uncompetitive inhibition. 6-Thiosucrose was a substrate for both enzymes to produce acceptor products when acceptors were present. GTF-I synthesized de novo a water-insoluble, (1----3)-6-thio-alpha-D-glucan from 6-thiosucrose.

Binding, Competitive↗