Anti-amnesic effect of enzyme extracted stevioside from Stevia rebaudiana

The increasing consumption of sucrose has resulted in several nutritional and medicinal problems, including obesity. There is an alarming rise in the prevalence of obesity, type 2 diabetes mellitus, and metabolic syndrome in children and adults around the world, partly related to increasing availability of energy-dense, high-calorie foods, and perhaps to increased consumption of sugar and particularly fructose sweetened beverages. Therefore, low calorie sweeteners are urgently required to substitute table sugar.

Stevioside, a diterpene glycoside, is well known for its intense sweetness and is used as a non-caloric sweetener. Its potential widespread use requires an easy and effective extraction method. Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase, pectinase and hemicellulase using various parameters such as concentration of enzyme, incubation time and temperature was optimized. The extraction conditions were further optimized using response surface methodology (RSM). Under the optimized conditions, the experimental values were in close agreement with predicted model and resulted in a three times yield enhancement of stevioside.

Various studies have revealed that in addition to sweetening nature of stevisoide, it exerts beneficial effects including antihypertensive, anti-hyperglycemic, anti-human rotavirus, antioxidant, anti-inflammatory and antitumor actions. Its anti-amnesic potential remains to be explored, therefore the present study has been undertaken to investigate the beneficial effect of stevioside in memory deficit of rats employing scopolamine induced amnesia as an animal model.

Significance: Stevia is gaining significance in different parts of the world and is expected to develop into a major source of high potency sweetener for the growing natural food market. There is a strong possibility that Stevia sweeteners could replace aspartame in some diet variants. In addition, Stevia is expected to be used as a part substitute for sugar and also used in combination with other artificial sweeteners in the emerging phase of life cycle.

Parameters important in fabricating enzyme electrodes using self-assembled monolayers of alkanethiols

The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.

Conducting polymer enzyme alloys : electromaterials exhibiting direct electron transfer

Glucose oxidase (GOx) is an important enzyme with great potential application for enzymatic sensing of glucose, in implantable biofuel cells for powering of medical devices in vivo and for large-scale biofuel cells for distributed energy generation. For these applications, immobilisation of GOx and direct transfer of electrons from the enzyme to an electrode material is required. This paper describes synthesis of conducting polymer (CP) structures in which GOx has been entrained such that direct electron transfer is possible between GOx and the CP. CP/enzyme composites prepared by other means show no evidence of such “wiring”. These materials therefore show promise for mediator-less electronic connection of GOx into easily produced electrodes for biosensing or biofuel cell applications.

Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His¹⁰⁸⁹ as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His¹⁰⁸⁹ to Ala or Leu. At pH 7.5, with Ang I as substrate, k_cat/K_m values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k_cat) with minor effects on ground state substrate binding (K_m). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His¹⁰⁸⁹ --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His¹⁰⁸⁹ side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His¹⁰⁸⁹ on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.

Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn²+ metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl−, but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl− dependence. Cl−dependence is also lost when the equivalent Arg in the N-domain, Arg⁵⁰⁰, is substituted with Gln. The Arg¹⁰⁹⁸ to Lys substitution reduced Cl− binding affinity by ∼100-fold. In the absence of Cl−, substrate binding affinity (1/K m) of and catalytic efficiency (k cat/K m) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg¹⁰⁹⁸ to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl−]. The Arg¹⁰⁹⁸ to Gln substitution also eliminates Cl− dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl−. These findings indicate that: 1) Arg¹⁰⁹⁸ is a critical residue of the C-domain Cl−-binding site and 2) a basic side chain is necessary for Cl− dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl−-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl− are much more important for the hydrolysis of short substrates than for Ang I. Since Cl− concentrations are saturating under physiological conditions and Arg¹⁰⁹⁸ is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl−-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.

Replacement of RNA hairpins by in vitro selected tetranucleotides

An in vitro selection method based on the autolytic cleavage of yeast tRNAPhe by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucieotide sequences allow proper folding of the rest of the tRNA molecule, wheras others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNAPhe and the anticodon hairpin of E.coli tRNAPhe without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally non essential hairpins in any RNA.

Antioxidant enzyme activities of iron-saturated bovine lactoferrin (Fe-bLf) in human gut epithelial cells under oxidative stress

Chemoprevention by dietary constituents in the form of functional food has emerged as a novel approach to control inflammatory diseases and cancers. Recently we reported for the first time that iron content is a critical determinant in the anti-tumour activity of bovine milk lactoferrin (bLf). We therefore wanted to evaluate the chemo-preventative efficacy of Apo-bLF and 100% iron-saturated bLF (Fe-bLF) on hydrogen peroxide (H2O 2)-induced colon carcinogenesis, and their influence on antioxidant enzyme activities within colon carcinogenesis. This was undertaken through observing how oxidative stress induced by H2O2 alters antioxidant enzyme activity within HT29 colon cancer cells, and then observing changes in this activity by treatments with the different antioxidants ascorbic acid (AA), Apo-bLF and Fe-bLF. All antioxidant enzymes (catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT) and superoxide dismutase (SOD)) appeared to be increased within HT29 cells, even prior to H2O2 exposure, and all enzymes showed significant decreased activity when cells were treated with the antioxidants AA, Apo-bLF or Fe-bLF, with or without H2O2 exposure. The results indicate that all three antioxidants have the ability to scavenge ROS, lower antioxidant enzyme activities within already excited states, and possibly allow colon cancer cells to be overcome by oxidative stress that would normally be prevented, perhaps leading to damage and potential apoptosis of the cancer cells. In conclusion, the anti-oxidative effects of Apo-bLF and Fe-bLf studied for the first time, show dynamic changes that may allow for necessary protection from imbalanced oxidative conditions, and potential at reducing the ability of cancer cells to protect themselves from oxidative stress states.

Phenylalanine-544 plays a key role in substrate and inhibitor binding by providing a hydrophobic packing point at the active site of insulin-regulated aminopeptidase

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn2+ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn2+ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the Ki in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.