Effect of uncouplers on "downhill" beta-galactoside transport in energy-depleted cells of Escherichia coli.
(9/86)
Galactoside permease-containing cells of Escherichia coli can be depleted of energy reserves so that the "downhill" cellular hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) no longer takes place. Treatment of such energy-depleted cells with proton-conducting agents such as carbonylcyanide m-chlorophenylhydrazone results in stimulation of ONPG transport. The same agents lower transport of non-energy-depleted cells towards the same levels that result from stimulation of the energy depleted cells. Of course, these agents prevent "uphill" accumulation against a concentration gradient under all conditions. Since uncouplers allow normal and energy-depleted cells to assume the same facilitated transport capability, these results lend support to the chemiosmotic hypothesis of Mitchell that comigration of charge is necessary for the transport of neutral galactosides. Our results imply that a potential favorable to transport is maintained by metabolism in non-energy-depleted cells, whereas an unfavorable potential is developed in the initial instant of time when energy-depleted cells are given ONPG. (+info)
Effect of dichlorodifluoromethane on the appearance, viability, and integrity of Escherichia coli.
(10/86)
Cultures of Escherichia coli H52 were treated with liquid dichlorodifluoromethane (fluorocarbon-12 [f-12]) for 2 h at 22 C and then examined microscopically. Treated cells tended to clump, and their cytoplasms were generally less dense and less uniform in appearance than those of control cells. E. coli ML30 was exposed to f-12 at a concentration of 1.25 X saturation for times up to 1,200 min at 22 C. Cells were examined for changes in viability (plate count), permeability (as measured by exit of alpha-[14-C]methylglucoside or uptake of omicron-nitrophenyl-beta-D-galactopyranoside), release of compounds absorbing at 260 nm, and lysis (changes in absorbance at 420 nm). Large losses of alpha-methylglucoside and of percentage of viability occurred after brief exposure to f-12. Release of compounds absorbing at 260 nm occurred more slowly than the aforementioned events, possibly because these molecules are larger than alpha-methylglucoside. During 1,200-min exposure to f-12, the number of survivors decreased from 10-9 to 10-4 organisms/ml, the loss of compounds absorbing at 260 nm amounted to 50 percent, and 32 percent lysis occurred. Most of these changes occurred during the first 300 min of treatment. Loss of alpha-methylglucoside was almost complete after 1-min exposure to f-12. These results suggest that death of the cell involves several stages, with a change of permeability, occurring first, followed by leakage of compounds of increasing size and, finally, lysis. (+info)
Lactose metabolism involving phospho-beta-galactosidase in Klebsiella.
(11/86)
Klebsiella strain RE1755A is a Lac- Gal- mutant which has lost both of its lac operons, but possesses a gene specifying beta-galactosidase III, an enzyme which hydrolyzes o-nitrophenyl-beta-D-galactopyranoside but does not hydrolyze lactose. Selective pressure was applied to isolate mutants able to utilize lactose. The lactose-utilizing mutants obtained were shown to possess an unaltered beta-galactosidase III. Lactose utilization was shown to result from a pleiotropic mutation which also (i) permits galactose utilization and (ii) prevents induction of beta-galactosidase III synthesis by lactose. Evidence is presented suggesting that a phospho-beta-galactosidase enzyme is involved in lactose metabolism. (+info)
Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichiacoli.
(12/86)
A mechanistic model for lactose/H(+) symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H(+) is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H(+) to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302-->Ala and Arg-302-->Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H(+) movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H(+) translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover. (+info)
Rapid confirmation of Clostridium perfringens by using chromogenic and fluorogenic substrates.
(13/86)
The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life. (+info)
Galactosyl transfer catalyzed by thermostable beta-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus: kinetic studies of the reactions of galactosylated enzyme intermediates with a range of nucleophiles.
(14/86)
The transfer of a galactosyl group from an enzyme to a number of neutral primary alcohols, phenol and azide has been studied during the reactions at 80 degrees C of thermostable beta-glycosidases from Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl beta-D-galactopyranoside or lactose (4-O-beta-D-galactopyranosyl D-glucopyranose) as substrates. The rate constant ratios, k(Nu)/k(water), for partitioning of the galactosylated enzyme intermediates between reaction with nucleophiles (k(Nu), M(-1) s(-1)) and water (k(water), s(-1)) have been determined from the difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding interactions contribute approximately 8 kJ mol(-1) to the stabilization of the transition state for the reaction of galactosylated enzyme intermediates of Ss beta Gly and CelB with 1-butanol, compared to the transition state for the enzymatic reaction with methanol. The leaving group/nucleophile binding sites of Ss beta Gly and CelB appear about 0.8 times as hydrophobic as n-octanol. Values of k(Nu)/k(water) for reactions of galactosylated Ss beta Gly with ethanol and substituted derivatives of ethanol show no clear dependence on the pK(a) of the primary hydroxy group of these nucleophiles in the pK(a) range 12.4-16.0. The binding of phenol with the galactosylated enzyme intermediates of Ss beta Gly and CelB occurs in a form that is mainly nonproductive pertaining to beta-galactoside synthesis. Neither enzyme catalyzes galactosyl transfer to azide ion. A model is proposed for the interaction of neutral nucleophiles at an extended acceptor site of the galactosylated enzymes. (+info)
Insights into the functional architecture of the catalytic center of a maize beta-glucosidase Zm-p60.1.
(15/86)
The maize (Zea mays) beta-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-A resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K(m) of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k(cat) was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation. (+info)
Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate.
(16/86)
A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties. (+info)