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C F Brewer

Publications and source records attributed to C F Brewer.

At least 91 records · Page 5Linked to original sources
PubMed1983

Stoichiometry of manganese and calcium ion binding to concanavalin A.

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.

Calcium↗
PubMed1983

Kinetics of conformational transitions of demetalized Concanavalin A.

Demetalized Concanavalin A exists in two conformational states, known as locked (PL) and unlocked (P) [Brown et al., Biochemistry 16, 3883 (1977)]. The equilibrium ratio [PL]/[P] is 0.14 +/- 0.01 at 25 degrees C, pH 6.4 [Brown et al., Biochemistry 21, 465 (1982)]. We now report values of the rate constants for the P in equilibrium; k1 = (33 +/- 4 h)-1 and k-1 = (4.6 +/- 0.6 h)-1 for the P leads to PL and PL leads to P transitions, respectively, at 25 degrees C, pH 6.4. The experiments utilize the fact that saccharide binds to PL [Koenig et al., Biochemistry 17, 4251 (1978)], producing a time-dependent increase in the total concentration of locked forms at equilibrium, and use a new technique for measuring this concentration.

Chemical Phenomena↗
PubMed1983

Metal ion binding and conformational transitions in concanavalin A: a structure-function study.

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn2+ and Ca2+, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a "locked" form and an "unlocked" form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn2+ and Ca2+. They also demonstrated that Mn2+ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25 degrees C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin. Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (approximately 22 kcal M-1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied. We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn2+ and saccharide, and have determined both metal ion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrates↗
PubMed1983

Synthesis and 19F spectra of tetra-L-alanine analogs containing selectively incorporated 3-fluoro-L-alanine residues.

The synthesis of analogs of tetra-L-alanine containing 3-fluoro-L-alanine selectively incorporated at each position is described. The standard procedures in the literature used to couple L-alanine peptides together were often found to lead to undesired products, or elimination reactions when corresponding 3-fluoro-L-alanine peptide analogs were used. Several modified procedures have thus been developed for the synthesis of fluorine-substituted analogs. In addition, the pH-dependence of 19F n.m.r. spectra of 3-fluoro-L-alanine and the tetrapeptide analogs is presented.

Alanine↗
PubMed1983

13C n.m.r. study of L-alanine peptides.

The di-, tri-, and tetrapeptides of L-alanine have been studied in aqueous solution by 13C n.m.r. spectroscopy at 25 and 50 MHz. By using selectively 13C enriched analogs containing either 90% 13C methyl or carbonyl carbons and measurements as a function of pH, assignment of the chemical shifts of the peptides has been made. T1 and NOE measurements of the peptides in their cationic, anionic, and zwitterionic states have been recorded as a function of concentration. The results show considerable segmental motion along the backbone carbons of the peptides, with only small changes occurring in the dynamic motions of the peptides as their charge states are altered. The lack of concentration dependence of the chemical shift and T1 values, as well as the similarity of T1 values for individual peptides in the three charge states, indicate that the peptides do not self-associate in aqueous solution.

Alanine↗
PubMed1982

Factors determining steric course of enzymic glycosylation reactions: glycosyl transfer products formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol by alpha-glucosidases and an inverting exo-alpha-glucanase.

Glycosyl transfer products were formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) by purified alpha-glucosidases from Candida tropicalis and rice and by an inverting exo-alpha-glucanase (glucodextranase) from Arthrobacter globiformis. The products were structurally defined through 1H and 13C NMR (nuclear magnetic resonance) spectra of their crystalline per-O-acetates in comparison with those of authentic methyl 1-deoxy-alpha- and methyl 1-deoxy-beta-D-gluco-heptuloside. 1-Deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-heptenitol and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptulose were produced by both the Candida alpha-glucosidase and the glucodextranase; 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 5)- and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptuloses by the rice alpha-glucosidase. These results, together with our earlier findings of sterospecific hydration of heptenitol catalyzed by the same enzymes [Hehre, E. J., Brewer, C. F., Uchiyama, T., Schlesselmann, P., & Lehmann, J. (1980) Biochemistry 19, 3557-3564], show the inadequacy of the long-accepted notion that carbohydrase-catalyzed reactions always lead to retention (or always lead to inversion) of substrate configuration. In particular, the finding that glucodextranase forms transfer products of alpha configuration and a hydration product of beta configuration from the same substrate provides a clear example of the functioning of acceptors rather than donor substrates in selecting the steric course of reactions catalyzed by a glycosylase. The circumstances under which acceptor cosubstrates might be expected to show this significant effect are discussed. The opportunity presumably would exist whenever carbonium ion mediated reactions are catalyzed by glycosylases that provide oppositely oriented approaches of different acceptors to the catalytic center.

Arthrobacter↗
PubMed1982

Trehalase: stereocomplementary hydrolytic and glucosyl transfer reactions with alpha- and beta-D-glucosyl fluoride.

A new understanding has been obtained of the catalytic capabilities of trehalase, an enzyme heretofore held to be strictly specific for hydrolyzing alpha, alpha-trehalose and devoid of transglycosylative ability. Highly purified rabbit renal cortical trehalase and a partly purified Candida tropicalis yeast trehalase were found to utilize both alpha- and beta-D-glucosyl fluoride as substrates. In each case, the reactions were competitively inhibited by alpha, alpha-trehalose. Both enzymes catalyzed rapid hydrolysis of alpha-D-glucosyl fluoride to form beta-D-glucose (also, of alpha, alpha-trehalose to form equimolar alpha- and beta-D-glucose). In addition, digests of beta-D-glucosyl fluoride plus alpha-D-[14C]-glucopyranose with either trehalase (but not controls of enzyme with alpha-D-[14C]glucopyranose alone) yielded small amounts of radioactive trehalose (alpha-D-glucopyranosyl alpha-D-[14C]glucopyranoside) which does not accumulate since it is rapidly hydrolyzed. Trehalase thus catalyzes two stereocomplementary types of glycosylation reactions: (I) alpha-D-glucosyl fluoride (or alpha, alpha-trehalose) + H2O leads to beta-D-glucose + HF (or alpha-D-glucose); (II) beta-D-glucosyl fluoride + alpha-D-glucopyranose leads to alpha, alpha-trehalose + HF. Such behavior shows that the catalytic groups of trehalase, as recently found for other glycosylases, are functionally flexible. The results illustrate the inadequacy of conventional views of carbohydrase specificity and the rigor, as a basic guiding principle, of the concept that glycoside hydrolases and glycosyltransferases form a class of glycosylases effecting glycosyl/proton interchange.

Animals↗
PubMed1982

Synthesis of the diastereoisomers of 1,2-dipalmitoyl-sn-glycero-3-thiophosphorylethanolamine and their stereospecific hydrolysis by phospholipases A2 and C.

A convenient three-step synthesis of the phosphorothioate analogue of phosphatidylethanolamine is described. The reaction pathway involves the conversion of a 1,2-diacyl-sn-glycerol to its corresponding thiophosphoric acid dichloride by using PSCl3 in the presence of a tertiary base. Treatment of the dichloride with ethanolamine results in the formation of a cyclic thiophosphoramidate which, upon acidification, undergoes P--N cleavage, giving rise to 1,2-diacyl-sn-glycero-3-thiophosphorylethanolamine. 31P NMR reveals that both diastereoisomers are present in equivalent amounts. It is not possible, however, to separate the two isomers by high-pressure liquid chromatography. 31P NMR amd high-pressure liquid chromatography are used to show that phospholipases A2 and C exhibit absolute and opposite stereoselectivity in the hydrolysis of the pair of diastereoisomers.

Bacillus cereus↗
PubMed1982

Conformational equilibrium of demetalized concanavalin A.

Concanavalin A (Con A) is known to exist in two conformations [Brown, R. D., III, Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883-3896] that differ in their metal ion and saccharide binding properties. The conformation that binds metal ions tightly, and which is associated with saccharide binding, has been designated as "locked" and that which binds metal ions only weakly as "unlocked". In the presence of excess metal ions, such as Mn2+ and Ca2+, essentially 100% of the protein is in the locked conformation. The scheme proposed to explain these effects [Koenig, S. H., Brewer, C. F., & Brown, R. D., III (1978) Biochemistry 17, 4251-4260] predicts an equilibrium between these conformations for the apoprotein. By monitoring the solvent proton relaxation dispersion as equimolar concentrations of Mn2+ and Ca2+ are titrated, at 5 degrees C, into an apo-Con A solution that had been equilibrated at 25 degrees C, we find that 12.5% of the apoprotein is in the locked conformation, corresponding to an energy separation of 1.2 kcal mol-1. We also show that these conformations can be separated by column chromatography at 5 degrees C and that the 100% unlocked form prepared in this way returns to the expected equilibrium mixture when kept at 25 degrees C.

Apoproteins↗
PubMed1982

Solution structure of 5-keto-D-fructose: relevance to the specificity of hexose kinases.

5-Keto-D-fructose (5KF) is isolated from cultures of Gluconobacter cerinus growing on D-fructose as the sole carbon source. 5KF is a substrate for hexokinase, fructokinase, and several polyol dehydrogenases. 1H and 13C nuclear magnetic resonance studies show that 5KF exists in different forms in anhydrous dimethyl-d6 sulfoxide and D2O. In dimethyl-d6 sulfoxide, 5KF exists as a spirane dimer with linked furanose and pyranose rings, similar to the structure reported for crystalline 5KF [Hassen, L., Hordvik, A., & Hove, R. (1976) J. Chem. Soc., Chem. Commun., 572-. In D2O, 5KF exists predominantly (greater than 95%) in a beta-pyranose form with the 5-keto group hydrated to form a gem-diol. 13C--1H coupling patterns, 13C relaxation measurements, and 13C deuterium-induced differential isotope shifts confirm this structure of 5KF. The phosphorylation of 5KF by fructokinase can be accounted for by an approximately 2% proportion of the beta-furanose form in solution at 25 degrees C. Both the beta-pyranose and beta-furanose forms of 5KF are proposed to be substrates for yeast hexokinase.

Deuterium↗
PubMed1981

Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride.

Rhizopus niveus glucoamylase and Arthrobacter globiformis glucodextranase, which catalyze the hydrolysis of starch and dextrans, respectively, to form D-glucose of inverted (beta) configuration, were found to convert both alpha- and beta-D-glucosyl fluoride to beta-D-glucose and hydrogen fluoride. Each enzyme directly hydrolyzes alpha-D-glucosyl fluoride but utilizes th beta-anomer in reactions that require 2 molecules of substrate and yield glucosyl transfer products which are then rapidly hydrolyzed to form beta-D-glucose. Various D-glucopyranosyl compounds serve as acceptors for such reactions. Mixtures of beta-D-glucosyl fluoride and methyl-alpha-D-glucopyranoside[14C], incubated with either enzyme, yielded both methyl-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-[14C]glucopyranoside and methyl-alpha-D-glucopyranosyl-(1 leads to 6)-alpha-D-[14C]glucopyranoside. Glucoamylase produced more of the alpha-maltoside; glucodextranase produced more of the alpha-isomaltoside. Thus, both "exo-alpha-glucan hydrolases" emerge as glucosylases that catalyze stereospecifically complementary hydrolytic and transglucosylative reactions with glucosyl donors of opposite configuration. These reactions not only provide a new view of the catalytic capabilities of these supposedly strict hydrolases; they also furnish a basis for defining a detailed mechanism for catalysis. Present results, together with those of several recent studies from this laboratory (especially similar findings obtained with beta-amylase acting on alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950), provide strong new evidence for the functional flexibility of the catalytic groups of carbohydrases.

Arthrobacter↗
PubMed1981

Chemical synthesis of radiolabeled bleomycin A2 and its binding to DNA.

A method for the preparation of biologically active [3H]- and [13C]bleomycin A2 is described. Demethyl Cu(II):bleomycin A2, isolated after pyrolysis of Cu(II):bleomycin A2, was methylated with either [3H]-or [13C]methyl iodide, which resulted in Cu(II):bleomycin A2 labeled in the dimethylsulfonium moiety. Copper was removed by treatment with dithizone in chloroform, and structures were verified by thin-layer chromatography and 1H and 13C nuclear magnetic resonance spectroscopy. Copper-free [3H]-and [13C]bleomycin A2 are active in the degradation of DNA in vitro. Gel exclusion chromatograhy and equilibrium dialysis were used to determine the apparent equilibrium constants for binding of [3H]bleomycin A2 and Cu(II):[3H]bleomycin A2 to calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate. In 2.5 mM sodium phosphate buffer, pH 7.0, binding data obtained by gel filtration with calf thymus DNA reveal an apparent equilibrium constant for [3H]bleomycin A2 of 5.7 X 10(5)/mol and for Cu(II):[3H]bleomycin A2 of 3.9 X 10(5)/mol. One molecule of [3H]bleomycin A2 binds for every 3.7 base pairs in DNA, and one molecule of Cu(II):[3H]bleomycin A2 binds for every 2.8 base pairs in DNA. Analysis of binding data with calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate obtained by equilibrium dialysis reveals, in each instance, 2 types of binding sites for both the copper and metal-free form of the antibiotic. For those sites in calf thymus DNA with tighter binding affinity, the apparent equilibrium constant for [3H]bleomycin A2 was 6.8 X 10(5)/mol and for the Cu(II):[3H]bleomycin A2 complex, 4.4 X 10(5)/mol. As seen with calf thymus DNA, the affinity of [3H]bleomycin A2 is slightly greater than that of Cu(II):[3H]bleomycin A2 for the synthetic DNAs, although more of the copper form of the drug binds to these polymers.

Animals↗