Electrostatic control of enzyme reactions: The mechanism of inhibition of glucose oxidase by putrescine

The interaction of putrescine dihydrochloride with glucose oxidase is reported. At pH 7.65 glucose oxidase is strongly anionic (Z = ?80). The pK_a of an essential acidic group on the reduced form of the enzyme is extremely sensitive to ionic strength, as predicted by simple electrostatic theory [J. G. Voet, J. Coe, J. Epstein, V. Matossian, and T. Shipley (1981), Biochemistry, 20, 7182–7185]. Putrescine dihydrochloride was found to inhibit glucose oxidase at pH 7.65 at a constant ionic strength of 0.05. The kinetics do not obey simple competitive inhibition, however. The data can best be explained by a model in which change in the electrostatic potential of the enzyme on putrescine binding changes the observed pK_a of the essential acidic group. The pH dependence of putrescine inhibition supports this interpretation. At I = 0.05, 5 mM putrescine was found to change the pK_a of the essential acidic group from 7.6 to 7.1. The shift in the pK_a as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The K_a for putrescine to the active form of the enzyme was calculated to be 4.2 mm.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Influence of Acidic pH on Hydrogen and Acetate Production by an Electrosynthetic Microbiome

Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm² at −765 mV (0.065 mA/cm² sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m³/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m³/day formate, and 3.1 kg/m³/day acetate ( = 4.7 kg CO₂ captured).     

Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB from Thermotoga neapolitana

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high V_max value and low apparent K_m value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the V_max values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the K_m values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The K_i values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the K_i values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.     

Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars

Lactobacillus rhamnosus GG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose on L. rhamnosus GG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log₁₀-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with other Lactobacillus strains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhanced L. rhamnosus GG survival from 6.4 to 8 log₁₀ CFU ml⁻¹ in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition of N′,N′-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F₀F₁-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F₀F₁-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 × 10⁶-fold compared with the survival of the wild type (which had a viability of 8.02 log₁₀ CFU ml⁻¹). L. rhamnosus GG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log₁₀ CFU ml⁻¹ in the presence of glucose. The data indicate that glucose provides ATP to F₀F₁-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.     

Phanerochaete chrysosporium β-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate β-glucosidases and an extracellular β-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K_m for p-nitrophenyl-β-glucoside is 1.6 × 10⁻⁴ M; the K_i for glucose, a competitive inhibitor, is 5.0 × 10⁻⁴ M. The K_m for cellobiose is 5.3 × 10⁻⁴ M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K_m for p-nitrophenyl-β-glucoside is 1.1 × 10⁻⁴ M. The molecular weight of this enzyme is ~410,000. Both enzymes have an optimal temperature of 45°C and an E_act of 9.15 kcal (ca. 3.83 × 10⁴ J). The pH optima, however, were ~7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ⁷⁰ holoenzyme, which shows σ region 1.1 (σ_1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ_1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ_1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.     

Permeant Cations and Blockers Modulate pH Gating of ROMK Channels

External potassium (K) activates the inward rectifier ROMK (K_ir1.1) by altering the pH gating of the channel. The present study examines this link between external K and internal pH sensitivity using both the two-electrode voltage clamp and the perfused, cut-open Xenopus oocyte preparation. Elevating extracellular K from 1 mM to 10 mM to 100 mM activated ROMK channels by shifting their apparent pK_a from 7.2 ± 0.1 (n = 6) in 1 mM K, to 6.9 ± 0.02 (n = 5) in 10 mM K, and to 6.6 ± 0.03 (n = 5) in 100 mM K. At any given internal pH, the number of active ROMK channels is a saturating function of external [K]. Extracellular Cs (which blocks almost all inward K current) also stimulated outward ROMK conductance (at constant 1 mM external K) by shifting the apparent pK_a of ROMK from 7.2 ± 0.1 (n = 6) in 1 mM K to 6.8 ± 0.01 (n = 4) in 1 mM K + 104 mM Cs. Surprisingly, the binding and washout of the specific blocker, Tertiapin-Q, also activated ROMK in 1 mM K and caused a comparable shift in apparent pK_a. These results are interpreted in terms of both a three-state kinetic model and a two-gate structural model that is based on results with KcsA in which the selectivity filter can assume either a high or low K conformation. In this context, external K, Cs, and Tertiapin-Q activate ROMK by destabilizing the low-K (collapsed) configuration of the selectivity filter.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA)·2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T_m for triplex decreases with increasing pH value in the presence of neomycin, while the T_m for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′, respectively. (4) The specific heat capacity change (ΔC_p) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the ΔC_p ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive ΔC_p is observed, with a value of −98 cal/(mol K) at 100 mM KCl. ΔC_p is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC₅₀ (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove.     

High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus

The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (P_QRS), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to P_QRS had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred P_QRS activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD_Ad-CarG_Ad). CarD_Ad-CarG_Ad is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD_Ad, whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.     

Actions of a Proline Analogue,L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas'' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC₅₀ = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.     

The Effect of an Adding Histidine on Biological Activity and Stability of Pc-pis from Pseudosciaena crocea

Pc-pis is a novel piscidin-like antimicrobial polypeptide that was identified in Pseudosciaena crocea. Although active against most bacteria tested, Pc-pis was inactive against Aeromonas hydrophila and Pseudomonas aeruginosa. The Pc-pis analogue Pc-pis-His was designed by adding a histidine residue at the carboxyl terminal. Pc-pis-His demonstrated a more broad-spectrum and stronger antimicrobial activity against a representative set of microorganisms and more potent antiparasitic activity against Cryptocaryon irritans trophonts than Pc-pis. The stability assay revealed that Pc-pis-His was active against Staphylococcus aureus not only in acidic (pH 5.5–7.3) and relatively low concentration monovalent cation (0–160 mM NaCl) environments but also in alkaline (pH 7.5–9.5), divalent cation (1.25–160 mM MgCl₂ and 1.25–40 mM CaCl₂) and high concentration monovalent cation (320–2560 mM NaCl) environments, which indicates that the added histidine residue conferred better salt-, acid- and alkali-tolerance to Pc-pis-His. Pc-pis-His also possessed the desired heat-tolerance, which was reflected by the antimicrobial activity of the peptide after being boiled for 10–60 minutes. Hemolytic activity analysis revealed that Pc-pis-His at concentrations up to 6 µM exhibited no hemolysis against human erythrocytes, with 6 µM being a concentration that is highly active against most of the microorganisms tested, although the hemolytic activity of Pc-pis-His was enhanced compared to Pc-pis. These results provide a unique, reasonable basis for designing novel piscidins with potent, broad-spectrum and stable antimicrobial activity and new insight into the future development of piscidins as potential therapeutic agents against microbial and external protozoan parasite infections.     

Leukocyte beta-glucosidase in homozygotes and heterozygotes for Gaucher disease

Human leukocytes contain at least two isozymes of 4-methylumbelliferyl-β-glucosidase acting optimally at pH 4.0 and 4.8; in Gaucher disease, only the former is deficient. Brief exposure of the leukocyte homogenate to pH 4.0 at room temperature results in irreversible inactivation of the pH 4.8 activity, while the activity at pH 4.0 remains unaffected. The more acidic isozyme is stimulated four- to fivefold by 0.2% sodium taurodeoxycholate (TDC) with a shift in the pH optimum to 5.0. The less acidic isozyme is completely suppressed in the presence of this detergent. Both leukocyte isozymes appear to be membrane-bound since gel filtration of Sephadex G-200 produces only one peak of activity located at the void volume, unlike in liver and kidney where a second peak also can be demonstrated. Heat inactivation analysis indicated that in controls, assayed in the absence of detergent, pH 4.0 activity is more thermostable than pH 4.8 activity. However, in Gaucher disease, the residual β-glucosidase at pH 4.0 is just as thermolabile as the unaffected pH 4.8 activity. Heat inactivation of the enzyme in the presence of TDC resulted in rapid loss of activity, suggesting a direct effect of the bile salt on the configuration of the enzyme decreasing its thermal stability. In the absence of detergent, acid β-glucosidase shows two K_m's, one at 3.2 mM and another at 0.9 mM. In the presence of detergent, only the higher K_m at 3.3 mM is obtained. In patients with Gaucher disease and in obligate carriers, the K_m remains essentially unaffected while the V_max shows the expected deficiency.

A reliable and reproducible selective assay technique has been developed for the diagnosis of Gaucher disease homozygotes and obligate heterozygotes and for the carrier screening of individuals at risk for this inherited disorder. The efficacy of this technique has been demonstrated by studying the activity in 42 controls, 26 patients, 32 obligate heterozygotes, and 23 healthy relatives of patients with Gaucher disease.