Effect of modification of sulfhydryl groups in soybean beta-amylase on the interaction with substrate and inhibitors

Methyl 2,4-dinitrophenyl disulfide (MDPS) is shown to be an effective methanethiolating reagent for sulfhydryl groups in proteins via thiol-disulfide exchange reaction. It reacts with the two reactive sulfhydryl groups (SH1 and SH2) in soybean beta-amylase. A decrease of the enzymatic activity accompanies the methanethiolation of SH2. After complete methanethiolation of SH2, the modified enzyme still has 9% of the initial activity. Modification of SH2 with cyanide and iodoacetamide reduces the enzymatic activity to 65 and 2% of the initial activity, respectively. Apparently, the residual activity depends upon the size of the substituent at SH2. The modified enzymes still have the almost same Km values for amylopectin and Kd values for enzyme-maltose and enzyme-cyclohexaamylose complexes as the native enzyme. In contrast to maltose and cyclohexaamylose, the Kd value of the enzyme-glucose complex increases in the order of cyanide-, MDPS-, and iodoacetamide-modified enzymes, indicating that SH2 is located near the binding site of glucose. It is proposed from the subsite structure of soybean beta-amylase that the position of SH2 and the glucose binding site is around subsite 1, where the nonreducing ends of the substrate bind productively.     

Difference spectroscopic study of the interaction between soybean beta-amylase and substrate or substrate analogues

1. In order to investigate the interactions between soybean beta-amylase [EC 3.2.1.2] and ligands (maltotriose as substrate, and maltose and alpha- and beta-cyclodextrins as inhibitors for the hydrolysis of maltoheptaose), the difference spectra were measured at 25 degrees C and pH 5.4, in 0.05 M acetate buffer. Each difference spectrum produced by these ligands showed a clear peak at 292-293 nm due to a tryptophan residue. In addition to this peak, the spectra of alpha- and beta-cyclodextrins showed a specific peak at 298-299 nm, and that of maltotriose showed a shoulder at 298 nm. 2. From the concentration dependency of the difference molar extinction delta epsilon, at 292-293 nm or at 298-299 nm, the dissociation constant of the enzyme-ligand complex, Kd, was evaluated for maltotriose, and alpha- and beta-cyclodextrins. For each ligand, the Kd values obtained at these two wavelengths were in good agreement with Michaelis constant, Km, or the inhibitor constant, Ki. The Kd value for maltose obtained from the titration of delta epsilon at 292 nm was also in good agreement with Ki. 3. Maltose produced a hydrophobic change in the environment of the tryptophan residue, while the interactions of maltotriose, and alpha- and beta-cyclodextrins with this enzyme caused an electrostatic change in the vicinity of the tryptophan residue in addition to the hydrophobic change. Since the signal at 298-299 nm was not found in the difference spectrum of maltose, this signal may be due to a tryptophan residue different from that which produces the signal at 292-293 nm. If both the signals are due to the same tryptophan residue, we must conclude that some conformational change is caused in the enzyme active site by the ligand binding.     

Interaction of native and SH-modified beta-amylase of soybean with cyclohexadextrin and maltose

Cyclohexadextrin and maltose bound to soybean beta-amylase and affected the environments of tryptophan and tyrosine residues, producing characteristic difference spectra in the ultraviolet region. The difference spectrum produced by cyclohexadextrin, a competitive inhibitor, had peaks at 285, 292, and 299 nm, while that by maltose, a reaction product, had peaks at 285 and 292 nm and a small trough at around 300 nm. By using the peaks at 292 and 299 nm, the dissociation constants of enzyme-cyclohexadextrin and enzyme-maltose complexes were calculated to be 0.35 mM and 8.1 mM, respectively. The effects of modification of SH groups of beta-amylase on the interaction of the enzyme with these sugars were examined by using beta-amylase carboxymethylated at the SH1 site and the enzyme modified at SH1 and SH2 sites with iodoacetamide or with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The dissociation constants of the enzyme-cyclohexadextrin and enzyme-maltose complexes were not changed by the modification of these SH groups, but the modification of SH2, the so-called essential SH group of soybean beta-amylase, strongly affected the difference spectra produced by maltose. The spectrophotometric titration of beta-amylase by cyclohexadextrin in the presence of maltose showed that cyclohexadextrin and maltose bind to the enzyme competitively, regardless of the modification of SH2. These results indicated that SH2 is located near the binding site of cyclohexadextrin and maltose, but is not involved in the binding of these sugars.     

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven.     

Kinetic properties of hexokinase under near-physiological conditions. Relation to metabolic arrest in Artemia embryos during anoxia

Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propose that inhibition by glucose-6-P (Kis = 65 microM) occurs primarily by competing with ATP at the active site, resulting in the formation of the dead-end complex, enzyme-glucose-glucose-6-P. Versus glucose, inhibition by glucose-6-P is uncompetitive at pH 8.0 and noncompetitive at pH 6.8. Over a physiologically relevant pH range of 8.0 to 6.8 alterations in Km and Ki values do not account for the reduction in glucose phosphorylation, and no evidence suggests that Artemia hexokinase activity is modulated by reversible binding to intracellular structures. Total aluminum in the embryos is 4.01 +/- 0.36 micrograms/g dry weight, or, based upon tissue hydration, 72 microM. This concentration of aluminum dramatically reduces enzyme activity at pH values less than 7.2, even in the presence of physiological metal ion chelators (citrate, phosphate). When pH, aluminum, citrate, phosphate, substrates, and products were maintained at cellular levels measured under anoxia, we can account for a 90% inhibition of hexokinase relative to activity under control (aerobic) conditions.     

Manganese (II) And spin-labeled uridine 5'-diphosphate binding to bovine galactosyltransferase.

The kinetically observed Mn(II) activation as well as inhibition has been clarified for bovine galactosyltransferase. An electron spin resonance (ESR) titration of MnCl2 with galactosyltransferase alone at pH 8.0 clearly shows the existence of at least two metal ion binding sites with microscopic dissociation constants of 0.84 +/- 0.1 and 9.0 +/- 1.0 mM, respectively. The second site corresponds with either published kinetic constant for Mn(II) of 8.5 mM (inhibition) or 3.40 mM (activation). The contribution of the binary complex Mn(II)-UDPGal is of lesser significance, as concluded by its ESR measured Kdiss of 14.5 +/- 1.1 mM at pH 8.0. A spin-labeled inhibitor analog of UDPgalactose, UDP-4-O-(2,2,6,6-tetramethyl-4-piperidinyl-1-oxy), or UDP-R, was synthesized as a competitive inhibitor for UDPGal. It was shown from inhibition kinetics to be almost as potent an inhibitor as UDPGlu. The Ki values at pH 8.0 in the N-acetyllactosamine and lactose reactions were 0.38 +/- 0.04 and 0.63 +/- 0.06 mM, respectively, as compared with 0.10 +/- 0.01 and 0.094 +/- 0.009 mM for UDPGlu. An ESR titration of UDP-R with galactosyltransferase at pH 8.0 yielded direct physical dissociation constants of 0.40 +/- 0.07 and 0.53 +/- 0.08 mM in the absence and presence of alpha-lactalbumin, respectively. No other substrates (glucose of N-acetylglucosamine) nor Mn(II) were present.     

Inhibition of urease by cyclic beta-triketones and fluoride ions

Competitive inhibition of soybean urease by 11 cyclic beta-triketones was studied in aqueous solutions at pH 7.4 and 36 degrees C. This process was characterized quantitatively by the inhibition constant (Ki), which showed a strong dependence on the structure of organic chelating agents (nickel atoms in urease) and varied from 58.4 to 847 microM. Under similar conditions, the substrate analogue (hydroxyurea) acted as a weak urease inhibitor (Ki = 6.47 mM). At 20 degrees C, competitive inhibition of urease with the ligand of nickel atoms (fluoride anion) was pH-dependent. At pH 3.85-6.45, the value of Ki for the process ranged from 36.5 to 4060 microM. Three nontoxic cyclic beta-triketones with Ki values of 58.4, 71.4, and 88.0 microM (36 degrees C) were the most potent inhibitors of urease. Their efficacy was determined by the presence of three >C=O- groups in the molecule and minimum steric hindrances to binding with metal sites in soybean urease.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

Kinetic studies with myo-inositol monophosphatase from bovine brain

The kinetic properties of myo-inositol monophosphatase with different substrates were examined with respect to inhibition by fluoride, activation or inhibition by metal ions, pH profiles, and solvent isotope effects. F- is a competitive inhibitor versus 2'-AMP and glycerol 2-phosphate, but noncompetitive (Kis = Kii) versus DL-inositol 1-phosphate, all with Ki values of approximately 45 microM. Activation by Mg2+ follows sigmoid kinetics with Hill constants around 1.9, and random binding of substrate and metal ion. At high concentrations, Mg2+ acts as an uncompetitive inhibitor (Ki = 4.0 mM with DL-inositol 1-phosphate at pH 8.0 and 37 degrees C). Activation and inhibition constants, and consequently the optimal concentration of Mg2+, vary considerably with substrate structure and pH. Uncompetitive inhibition by Li+ and Mg2+ is mutually exclusive, suggesting a common binding site. Lithium binding decreases at low pH with a pK value of 6.4, and at high pH with a pK of 8.9, whereas magnesium inhibition depends on deprotonation with a pK of 8.3. The pH dependence of V suggests that two groups with pK values around 6.5 have to be deprotonated for catalysis. Solvent isotope effects on V and V/Km are greater than 2 and 1, respectively, regardless of the substrate, and proton inventories are linear. These results are consistent with a model where low concentrations of Mg2+ activate the enzyme by stabilizing the pentacoordinate phosphate intermediate. Li+ as well as Mg2+ at inhibiting concentrations bind to an additional site in the enzyme-substrate complex. Hydrolysis of the phosphate ester is rate limiting and facilitated by acid-base catalysis.     

Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.     

Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Crystal structure of a catalytic site mutant of beta-amylase from Bacillus cereus var. mycoides cocrystallized with maltopentaose

The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172 --> Ala), from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-type enzyme complexed with maltose were determined at 2.1 and 2.0 A resolution, respectively. Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed. All glucose residues adopted a relaxed (4)C(1) conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-amylase, but differed from that of G5 in E172A. When the substrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DeltaG(++), which is the difference in free energy between this state and the transition state.     

An ATP-modulated specific association of glyceraldehyde-3-phosphate dehydrogenase with human erythrocyte glucose transporter

Glyceraldehyde-3-phosphate dehydrogenase was found to bind in vitro to purified, human erythrocyte glucose transporter reconstituted into vesicles. Mild tryptic digestion of the glucose transporter totally inactivated the binding, suggesting that the cytoplasmic domain of the transporter is involved in the binding to glyceraldehyde-3-phosphate dehydrogenase. The binding was abolished in the presence of antisera raised against the purified glucose transporter, further supporting specificity of this interaction. The binding was reversible with a dissociation constant (Kd) of 3.3 x 10(-6) M and a total capacity (Bt) of approximately 30 nmol/mg of protein indicating a stoichiometry of one enzyme-tetramer per accessible transporter. The binding was sensitive to changes in pH showing an optimum at around pH 7.0. KCl and NaCl inhibited the binding in a simple dose-dependent manner with Ki of 40 and 20 mM, respectively. The binding was also inhibited by NAD+ with an estimated Ki of 3 mM. ATP, on the other hand, enhanced the binding by up to 3-fold in a dose-dependent manner with an apparent Ka of approximately 6 mM. The binding was not affected by D-glucose or cytochalasin B. The binding did not affect either the glucose or cytochalasin B in binding affinities or the transport activity of the transporter. However, the enzyme was inactivated totally upon binding to the transporter. Based on these findings, we suggest that a significant portion of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes exists as an inactive form via an ATP-dependent, reversible association with glucose transporter, and that this association may exert regulatory intervention on nucleotide metabolism in vitro.     

Effects of some chlorinated sugar derivatives on the hexose transport system of the blood/brain barrier.

The inhibition of D-glucose transport into brain by several hexose analogues has been investigated in adult anaesthetized rats. D-Glucose was transported with apparent Vmax. = 1.22 mumol/g per min, Km = 11.12 mM and Kd = 0.008 ml/g per min. 6-Chloro-6-deoxyglucose was transported with corresponding values of Vmax. = 1.33 mumol/g per min, Km = 5.5 mM and Kd = 0.0155 ml/g per min and inhibited D-glucose transport with apparent Ki = 3.01 mM. 6-Chloro-6-deoxymannose, 6-chloro-6-deoxygalactose and 6-tosyl-6-deoxygalactose also inhibited D-glucose transport, but 6-chloro-6-deoxyfructose was without effect. The results were consistent with a model for glucose transport at the blood/brain interface that involves a hydrophobic site on the transport protein at or near the 6-position of bound glucose.     

Effects of High Pressure on Glucose Transport in the Human Erythrocyte

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.     

The effect of pancreatic procolipase and colipase on pancreatic lipase activation

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.     

4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Purification and properties

An enzyme which catalyzes the transamination of 4-aminobutyrate with 2-oxoglutarate was purified 588-fold to homogeneity from Candida guilliermondii var. membranaefaciens, grown with 4-aminobutyrate as sole source of nitrogen. An apparent relative molecular mass of 107,000 was estimated by gel filtration. The enzyme was found to be a dimer made up of two subunits identical in molecular mass (Mr 55,000). The enzyme has a maximum activity in the pH range 7.8-8.0 and a temperature optimum of 45 degrees C. 2-Oxoglutarate protects the enzyme from heat inactivation better than pyridoxal 5'-phosphate. The absorption spectrum of the enzyme exhibits two maxima at 412 nm and 330 nm. The purified enzyme catalyzes the transamination of omega-amino acids; 4-aminobutyrate is the best amino donor and low activity is observed with beta-alanine. The Michaelis constants are 1.5 mM for 2-oxoglutarate and 2.3 mM for 4-aminobutyrate. Several amino acids, such as alpha,beta-alanine and 2-aminobutyrate, are inhibitors (Ki = 38.7 mM, Ki = 35.5 mM and Ki = 33.2 mM respectively). Propionic and butyric acids are also inhibitors (Ki = 3 mM and Ki = 2 mM).     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines. Molecular modeling of complexes with glucoamylase

The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.