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B Ganem

Publications and source records attributed to B Ganem.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification of two functionally different classes of exocellulases.

There are two classes of synergism in cellulase mixtures: synergism between endocellulases and exocellulases, and synergism between certain exocellulases. Exocellulases have been defined traditionally as releasing cellobiose from the nonreducing ends of cellulose, but this definition is inadequate to explain exo/exo synergism. Several recent reports indicate that some exocellulases are capable of hydrolyzing cellulose from the reducing end. The existence of two exocellulase classes with different specificities could provide an explanation for exo/exo synergism. In this paper, we report the substrate specificity of three Thermomonospora fusca (E3, E4, and E6) and two Trichoderma reesei (CBH I and CBH II) exocellulases on labeled cellooligosaccharides. We describe a new nonradioactive technique for determining substrate specificity, in which ion-spray mass spectrometry was used to analyze the products of enzymatic digests of cellopentaose labeled with 18O at the reducing end. Exocellulase reactivity was also investigated on cellopentaose labeled at the nonreducing end with 14C, and cellooligosaccharides reduced with NaBH4. The distribution of label in the reaction products supports the existence of two functional classes of exocellulases. One class (containing CBH I, E4, and E6) preferentially cleaves cellooligosaccharides from the reducing end, while the other (containing E3 and CBH II) preferentially cleaves from the nonreducing end. This classification of exocellulases is consistent with exo/exo synergism experiments, and with published cellulase crystallographic data.

Actinomycetales↗
PubMed1996

Site-directed mutagenesis of monofunctional chorismate mutase engineered from the E. coli P-protein.

Analysis of the active-site residues of a fully functional chorismate mutase representing the N-terminal 113 amino acids of the Escherichia coli P-protein suggests that Lys39 and Gln88 play critical roles in catalyzing the rearrangement of chorismate to prephenate. Five site-directed mutants at these positions have been constructed in which Lys39 was replaced with Arg, Asn, and Gln, and Gln88 was replaced with Arg and Glu. Although the Gln88Arg plasmid failed to produce detectable cross-reacting proteins in E. coli, the other four plasmids were expressed, and the mutant proteins purified to homogeneity. Their structures were similar to wild type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing a small deviation. In accordance with predictions, all mutations result in major loss of catalytic activity at pH 7.8. However, activity of the Gln88Glu mutant at pH 4.5 exceeded wild-type EcCM. Implications for the mechanism of mutase catalysis are discussed.

Bacterial Proteins↗
PubMed1996

Charge is the major discriminating factor for glutathione reductase versus trypanothione reductase inhibitors.

Benson et al. (Biochem. J. 1992, 286, 9) reported three novel competitive inhibitors of trypanothione reductase (TR), which were selected to complement a hydrophobic region identified on the TR structure which was not present on human glutathione reductase (hGR). Benson et al. also noted that chlorpromazine, a tricyclic antidepressant known to have trypanocidal activity, was an inhibitor of TR. Here we show that chlorpromazine is a competitive inhibitor of TRs from Crithidia fasciculata (Ki = 14 microM) and Trypanosoma cruzi (Ki = 10 microM), but the drug binds > 50-fold more weakly (Ki = 762 microM) to hGR. Analogues of chlorpromazine differing in the length of the side chain carrying the positively charged R-group are also selective TR inhibitors whereas, a tricyclic structure carrying a negatively charged side chain is a competitive inhibitor with selectivity for hGR (K(hGR)i = 165 microM vs. K(TR)i = 1400 microM). This finding suggests that simple charge characteristics, rather than differences in hydrophobicity, may account for a significant portion of the selectivity of this series of inhibitors for these two enzymes. Electrostatic analysis of the structures of TR and hGR thus provides a rationale for these results, and offers a new principle for inhibitor design. The principle gains further support from the observation that all known tricyclic competitive inhibitors of TR are positively charged. In order to investigate the in vivo relevance of our findings we have examined the effect of chlorpromazine and its negatively charged analogue on the growth of C. fasciculata parasites. Consistent with our kinetics, chlorpromazine (50 microM) inhibited the growth of parasites by 50%, while no measurable decrease in parasite growth rate was noted in the presence of the negatively charged inhibitor (400 microM). Furthermore, the highly similar inhibitory profiles of C. fasciculata TR and T. cruzi TR suggest that drug-design studies using the structurally better-studied C. fasciculata TR are also relevant to the human pathogen T. cruzi.

Animals↗
PubMed1995

Kukoamine A and other hydrophobic acylpolyamines: potent and selective inhibitors of Crithidia fasciculata trypanothione reductase.

The enzyme trypanothione reductase (TR), together with its substrate, the glutathione-spermidine conjugate trypanothione, plays an essential role in protecting parasitic trypanosomatids against oxidative stress and is a target for drug design. Here we show that a naturally occurring spermine derivative, the antihypertensive agent kukoamine A [N1N12-bis(dihydrocaffeoyl)-spermine] inhibits TR as a mixed inhibitor (Ki = 1.8 microM, Kii = 13 microM). Kukoamine shows no significant inhibition of human glutathione reductase (Ki > 10 mM) and thus provides a novel selective drug lead. The corresponding N1N8-bis(dihydrocaffeoyl)spermidine derivative was synthesized and acted as a purely competitive inhibitor with Ki = 7.5 microM. A series of mono- and di-acylated spermines and spermidines were synthesized to gain an insight into the effect of polyamine chain length, the nature and position of the acyl substituent and the importance of conformational mobility. These compounds inhibited TR with Ki values ranging from 11 to 607 microM.

Animals↗
PubMed1995

Kinetic monitoring of enzymatic reactions in real time by quantitative high-performance liquid chromatography-mass spectrometry.

The study of enzyme kinetics under steady-state conditions represents a common and very useful method for investigating the mechanisms of enzymatic reactions. We report the use of mass spectrometry (MS) coupled with HPLC for the kinetic analysis of enzymatic reactions in real time. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A (RNase A) and the substrate-specific hydrolysis of lactose with beta-galactosidase can be monitored using ion-spray (pneumatically assisted electrospray) mass spectrometry as a sensitive and specific detector for the native substrates. The resulting data can be used to calculate both KM and Vmax for each system. Kinetic parameters obtained for RNase A and beta-galactosidase paralleled those obtained by conventional techniques. These findings suggest the possibility of developing alternative techniques, based on mass spectrometric detection, for performing kinetic analyses of enzymatic processes where no simple spectrophotometric assay is feasible. In addition to enabling the determination of kinetic parameters for authentic substrates, and not chromogenic analogs, such assays would also be useful in situations where very high sensitivity and specificity are desired.

Animals↗
PubMed1995

New insight into the catalytic mechanism of chorismate mutases from structural studies.

Chorismate mutase catalyzes the rearrangement of chorismic acid to prephenic acid, which is the first committed step in the biosynthesis of aromatic amino acids. Its catalytic mechanism has been much studied, but is poorly understood. Recent structural information on enzymes from two species, and on an antibody that catalyzes the same reaction, has shed new light on this topic.

Animals↗
PubMed1995

Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth.

Analogs and homologs of PDMP were synthesized, based on its structure (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol). This compound had previously been found to block the synthesis of GlcCer (glucosylceramide). Increasing the acyl chain length from 10 to 16 carbon atoms greatly enhanced the efficacy of the enzyme inhibitor, as did the use of a less polar cyclic amine, especially a pyrrolidine instead of a morpholine ring. Replacement of the phenyl ring by a chain corresponding to sphingosine also yielded a strongly inhibitory material. By using a chiral synthetic route, we showed that the isomers active against GlcCer synthase had the R,R-(D-threo)-configuration. However, strong inhibition of the growth of human cancer cells in plastico was produced by both the threo and erythro racemic compounds, showing involvement of an additional factor (beyond simple depletion of cell glycosphingolipids by blockage of GlcCer synthesis). The growth arresting effects could be correlated with increases in cellular ceramide and diglyceride levels. The aliphatic pyrrolidino compound was strongly inhibitory toward the glucosyltransferase and produced almost complete depletion of glycolipids, but did not inhibit growth or cause an accumulation of ceramide. Attempts were made to see whether the differences in growth effects could be attributed to the influence of the inhibitors on related enzymes (ceramide and sphingomyelin synthase and ceramidase and sphingomyelinase). While some stimulation of enzyme activity was noted, particularly at high inhibitor concentrations (50 microM), these findings did not explain the differing effects of the different inhibitors. The best inhibitors of GlcCer synthase compared favorably in efficacy with some cancer chemotherapeutic drugs in current use when tested with a battery of human cancer cells.

Animals↗
PubMed1994

Amidrazone analogues of D-ribofuranose as transition-state inhibitors of nucleoside hydrolase.

The transition state of inosine during hydrolysis by nucleoside hydrolase has been characterized by kinetic isotope effects, bond-energy/bond-order vibrational analysis, and molecular electrostatic potential surface calculations [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795; Horenstein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The heterocyclic base is protonated and the anomeric carbon of the ribofuranosyl ring is flattened to form a transition-state with extensive oxocarbenium ion character. With their delocalized charge and flattened structures, amidrazone analogues of D-ribofuranose provide both geometric and electronic mimics of the ribosyl group at the transition-state of nucleoside hydrolase. A family of riboamidrazones was synthesized with H, phenyl, and p-nitrophenyl N-substituents. The analogues were competitive inhibitors with respect to inosine and gave Ki values of 10(-5), 2 x 10(-7), and 1 x 10(-8) M, respectively. (p-Nitrophenyl)riboamidrazone exhibited slow-onset, tight-binding inhibition, with an overall dissociation constant of 2 x 10(-9) M. The binding is reversible with an off-rate of 3 x 10(-3) s-1. Tight binding can be attributed to the close spatial match between the molecular geometry of (p-nitrophenyl)riboamidrazone and the transition-state stabilized by nucleoside hydrolase. The favorable binding interactions of the (p-nitrophenyl)riboamidrazone include oxocarbenium ion mimicry, isosteric ribosyl hydroxyls, and hydrophobic and H-bonding interactions at the nitrophenyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Studies of non-covalent interactions of actinomycin D with single-stranded oligodeoxynucleotides by ion spray mass spectrometry and tandem mass spectrometry.

Non-covalent binding of actinomycin D (AMD) to single-stranded DNA (ssDNA) was observed by ion spray mass spectrometry. Interactions between AMD and different sequences of non-self-complementary oligodeoxynucleotides were investigated as a function of pH. Non-covalent AMD/ssDNA complexes disappeared at low pH, and no ssDNA complexes were detected with rifampicin (RP). Moreover, the different binding affinities between AMD and ssDNA with and without 5'G-C3' or 5'C-G3' base sequences were also demonstrated using ion spray mass spectrometry. Additional support for the non-covalent nature of binding in AMD/ssDNA complexes was obtained from tandem mass spectrometry.

Base Sequence↗
PubMed1993

Mass spectrometric investigations of drug-receptor interactions.

Mass spectrometric (MS) detection of biologically important noncovalent complexes is reviewed within the scope of on-going studies on this topic from this laboratory. The merits of ion spray MS and tandem MS are demonstrated in this area with examples that show systemic studies of several receptor-ligand, enzyme-substrate, and enzyme-product complexes associated with both an established well-characterized biological system (lysozyme hydrolysis of N-acetylglucosamine) and an important modern receptor-ligand complex (FKBP-FK-506 and FKBP-rapamycin). These examples and recent applications of negative ion MS and MS/MS with DNA adducts, heme-globin complexes, and antibiotic-binding proteins suggest an exciting new area of applications for MS that have not been previously available to investigators.

Animals↗
PubMed1992

Cytostasis induced in L1210 murine leukaemia cells by the S-adenosyl-L-methionine decarboxylase inhibitor 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine may be due to hypusine depletion.

The effects of inhibition of the capacity to form spermidine and spermine on cell growth were investigated using murine leukaemia L1210 cells and 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811, AbeAdo), an enzyme-activated irreversible inhibitor of S-adenosyl-L-methionine decarboxylase. Putrescine levels were increased 80-fold, and spermidine and spermine levels were greatly reduced after a 3-day exposure to a maximally inhibitory dose of 10 microM-AbeAdo. Addition of AbeAdo to the culture medium inhibited the growth of L1210 cells measured 3 days later in a dose-dependent manner, but, even at a dose of 10 microM, which was maximally effective, exposure to AbeAdo was not immediately cytostatic. However, the growth rate of L1210 cells chronically exposed to 10 microM-AbeAdo declined steadily until day 12, when the cells stopped growing. L1210 cells exposed to AbeAdo for 12 days could not be rescued from cytostasis by removal of the drug from the culture, but could be rescued by exposure to exogenous spermidine or spermine, indicating that the growth-inhibitory effects of AbeAdo were a result of spermidine and/or spermine depletion. It is suggested that elevated intracellular putrescine in AbeAdo-treated cells sustained limited growth in the absence of physiological levels of spermidine and spermine until certain critical and specific physiological role(s) fulfilled by spermidine (and/or spermine) became deficient resulting in cytostasis. N-(3-Aminopropyl)-1,4-diamino-cis-but-2-ene, a spermidine analogue that is a substrate for deoxyhypusine synthase, was able to mimic the effects of spermidine in reversing AbeAdo-induced cytostasis. Spermidine analogues such as 5,5-dimethylspermidine, which are not substrates for deoxyhypusine synthase, were not active in this way. These results provide evidence that the formation of hypusine in the protein-synthesis initiation factor eIF-5A may be a critical role of spermidine essential for cell growth.

Adenosylmethionine Decarboxylase↗
PubMed1992

D-mannonolactam amidrazone. A new mannosidase inhibitor that also inhibits the endoplasmic reticulum or cytoplasmic alpha-mannosidase.

The amidrazone of D-mannonolactam (see compound 5, Fig. 1) was synthesized chemically as a mimic of the mannopyranosyl cation and tested as a potential inhibitor of mannosidases. In this study compound 5 is shown to be a more general mannosidase inhibitor than other currently known compounds and exhibits properties not previously observed with any other mannosidase inhibitors. Thus D-mannonolactam amidrazone not only inhibits the Golgi mannosidase I (IC50 = 4 microM) and mannosidase II (IC50 = 90-100 nM), but it is the first inhibitor that has been shown to be a potent inhibitor of the soluble or endoplasmic reticulum alpha-mannosidase (IC50 = 1 microM). This compound also inhibited the aryl-mannosidases regardless of anomeric configuration although it was much more effective on enzymes recognizing alpha-linked mannose, i.e. jack bean and mung bean alpha-mannosidases (IC50 = 400 nM) as compared with fungal beta-mannosidase (IC50 = 150 microM). Mannonoamidrazone was tested in animal cell cultures using influenza virus-infected Madin-Darby canine kidney cells as a model system, and was found to prevent almost completely the formation of complex types of N-linked oligosaccharides with the formation of about equal amounts of Man9(GlcNAc)2 and Man8(GlcNAc)2 structures. Thus D-mannonolactam amidrazone is a potent but broad spectrum mannosidase inhibitor whose structure and properties should provide valuable insight into the design of other useful glycosidase inhibitors.

Animals↗
PubMed1991

Role of unsaturated derivatives of spermidine as substrates for spermine synthase and in supporting growth of SV-3T3 cells.

Synthetic unsaturated analogues of the natural polyamine were examined as possible substrates for spermine synthase and as replacements for spermidine in supporting the growth of SV-3T3 cells. It was found that N-(3-aminopropyl)-1,4-diamino-cis-but-2-ene [the cis isomer of the alkene analogue of spermidine] was a good substrate for spermine synthase, but that the trans isomer [N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and the alkene analogue [N-(3-aminopropyl)-1,4-diaminobut-2-yne] were not substrates. These results provide the first demonstration of stereospecificity in the spermine synthase reaction. All three of the unsaturated spermidine analogues described above and the cis-alkene analogue of spermine [N1N4-bis-(3-aminopropyl)-1,4-diamino-cis-but-2-ene] were able to support the growth of SV-3T3 cells that were prevented from the endogenous synthesis of spermidine by treatment with alpha-difluoromethylornithine. Since N-(3-aminopropyl)-1,4-diamino-trans-but-2-ene] and N-(3-aminopropyl)-1,4-diaminobut-2-yne were not converted into a spermine derivative, it is apparent that this conversion is not needed for the stimulation of growth. However, since N1N4-bis-(3-aminopropyl)-1,4-diamino-cis-but-2-ene was also able to support growth and was not degraded to the spermidine derivative, it appears that either polyamine can be effective in this respect. All of the unsaturated analogues tested accumulated in the SV-3T3 cells to a much greater extent than spermidine itself. This indicates that these compounds are substrates for the polyamine transport system, but that they are less effective than the natural polyamines in the feedback regulation of this system.

Animals↗
PubMed1991

U-3'-BCIP: a chromogenic substrate for the detection of RNase A in recombinant DNA expression systems.

The synthesis of the bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5) chromogenic substrate uridine-3'-(5-bromo-4-chloroindol-3-yl)-phosphate (U-3'-BCIP) is described. RNase A catalyzes the hydrolysis of U-3'-BCIP to release a halogenated indol-3-ol that undergoes rapid aerobic oxidation to the dark blue 5,5'-dibromo-4,4'-dichloroindigo. Preliminary kinetic studies indicate that this compound may have practical use for assaying RNase A activity both in vitro and in vivo, e.g. in screening bacterial colonies for RNase A produced by recombinant DNA methods.

Animals↗
PubMed1990

Easy assembly of ligands for glycosidase affinity chromatography.

An improved, high-yield synthesis of the corresponding N-carboxypentyl derivatives of three iminoalditol glycosidase inhibitors has been developed for affinity chromatography enzyme purification. Reductive amination of 1-deoxynojirimycin (or its D-manno or D-galacto analogues) with methyl 5-formylvalerate and NaBH3CN at neutral pH afforted an aminoester which upon hydrolysis with aqueous 5% HCl gave the desired aminoacid in 97% overall yield. These amino acids could then be covalently attached using water-soluble carbodi-imide to 6-aminohexyl Sepharose 4B.

1-Deoxynojirimycin↗
PubMed1988

Studies of non-metabolizable polyamines that support growth of SV-3T3 cells depleted of natural polyamines by exposure to alpha-difluoromethylornithine.

A number of synthetic polyamine derivatives that included five achiral gem-dimethylspermidines and two analogous tetramethylated spermines were tested for their abilities to serve as substrates for enzymes metabolizing polyamines and for their capacities to substitute for the natural polyamines in cell growth. It was found that none of the compounds were effective substrates for spermine synthase, and only one, namely 8,8-dimethylspermidine, was a substrate for spermidine/spermine N1-acetyltransferase. However, all of the spermidine derivatives and 1,1,12,12-tetramethylspermine were able to support the growth of SV-3T3 cells in which endogenous polyamine synthesis was prevented by the addition of alpha-difluoromethylornithine. These results suggest that either spermidine or spermine can support cell growth without the need for metabolic interconversion. In contrast with the result with 1,1,12,12-tetramethylspermine, 3,3,10,10-tetramethylspermine did not restore growth of polyamine-depleted SV-3T3 cells. Comparison of the properties of these derivatives may prove valuable in understanding the physiological role of polyamines.

Acetyltransferases↗