Some physicochemical and rheological properties of starch isolated from avocado seeds.

Seeds from avocado (Persea americana Miller) fruit are a waste byproduct of fruit processing. Starch from avocado seed is a potential alternative starch source. Two different extraction solvents were used to isolate starch from avocado seeds, functional and rheological characteristics measured for these starches, and comparisons made to maize starch. Avocado seed powder was suspended in a solution containing 2 mM Tris, 7.5 mM NaCl and 80 mM NaHSO3 (solvent A) or sodium bisulphite solution (1500 ppm SO2, solvent B). Solvent type had no influence (p>0.05) on starch properties. Amylose content was 15-16%. Gelatinization temperature range was 56-74 °C, peak temperature was 65.7 °C, and transition enthalpy was 11.4-11.6J/g. At 90 °C, solubility was 19-20%, swelling power 28-30 g water/g starch, and water absorption capacity was 22-24 g water/g starch. Pasting properties were initial temperature 72 °C; maximum viscosity 380-390 BU; breakdown -2 BU; consistency 200 BU; and setback 198 BU. Avocado seed starch dispersions (5% w/v) were characterized as viscoelastic systems, with G'>G″. Avocado seed starch has potential applications as a thickening and gelling agent in food systems, as a vehicle in pharmaceutical systems and an ingredient in biodegradable polymers for food packaging.

Effects of a buried cysteine-to-serine mutation on yeast triosephosphate isomerase structure and stability.

All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132-138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.

Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases.

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10⁻³ M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s⁻¹, and 1.2 +/- 0.105 × 104 M⁻¹s⁻¹, respectively. The L-asparaginase activity was reduced in the presence of Mn²âº, Zn²âº, Ca²âº, and Mg²âº metal ions for about 52% to 31%. In addition, we found that NH4⁺, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.

Crystal structure of Cu / Zn superoxide dismutase from Taenia solium reveals metal-mediated self-assembly.

Taenia solium is the cestode responsible for porcine and human cysticercosis. The ability of this parasite to establish itself in the host is related to its evasion of the immune response and its antioxidant defence system. The latter includes enzymes such as cytosolic Cu/Zn superoxide dismutase. In this article, we describe the crystal structure of a recombinant T. solium Cu/Zn superoxide dismutase, representing the first structure of a protein from this organism. This enzyme shows a different charge distribution at the entrance of the active channel when compared with human Cu/Zn superoxide dismutase, giving it interesting properties that may allow the design of specific inhibitors against this cestode. The overall topology is similar to other superoxide dismutase structures; however, there are several His and Glu residues on the surface of the protein that coordinate metal ions both intra- and intermolecularly. Interestingly, one of these ions, located on the ß2 strand, establishes a metal-mediated intermolecular ß-ß interaction, including a symmetry-related molecule. The factors responsible for the abnormal protein-protein interactions that lead to oligomerization are still unknown; however, high metal levels have been implicated in these phenomena, but exactly how they are involved remains unclear. The present results suggest that this structure could be useful as a model to explain an alternative mechanism of protein aggregation commonly observed in insoluble fibrillar deposits.

Chemical and pharmacological aspects of capsaicin.

Capsaicin is a unique alkaloid found primarily in the fruit of the Capsicum genus and is what provides its spicy flavor. Generally extracted directly from fruit, high demand has driven the use of established methods to increase production through extraction and characterization. Over time these methods have improved, usually be applying existing techniques in conjunction. An increasingly wide range of potential applications has increased interest in capsaicin. Especially compelling are the promising results of medical studies showing possible beneficial effects in many diseases. Capsaicin's pungency has limited its use in clinical trials to support its biological activity. Characterization and extraction/ synthesis of non-pungent analogues is in progress. A review is made of capsaicin research focusing mainly on its production, synthesis, characterization and pharmacology, including some of its main potential clinical uses in humans.

Binding thermodynamics of phosphorylated inhibitors to triosephosphate isomerase and the contribution of electrostatic interactions.

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (K(b), ΔG(b), ΔH(b), ΔS(b) and ΔC(p)) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔC(p) in a single experiment. We ruled out specific interactions of Na(+) and Cl(-) with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused K(b), ΔG(b) and ΔH(b) to become less favorable, while ΔS(b) became less unfavorable. From the variation of K(b) with I, we determined the electrostatic contribution of TIM-2PG and TIM-PGH to ΔG(b) at I=0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔH(b) compared to 2PG (by 19-24 kJ mol(-1) at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔC(p)s were negative, and less negative with increasing ionic strength. ΔC(p)s at I=0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM-2PG changed more with ionic strength than those for TIM-PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.

The conserved salt bridge linking two C-terminal beta/alpha units in homodimeric triosephosphate isomerase determines the folding rate of the monomer.

Triosephosphate isomerase (TIM), whose structure is archetypal of dimeric (beta/alpha)(8) barrels, has a conserved salt bridge (Arg189-Asp225 in yeast TIM) that connects the two C-terminal beta/alpha segments to rest of the monomer. We constructed the mutant D225Q, and studied its catalysis and stability in comparison with those of the wild-type enzyme. Replacement of Asp225 by Gln caused minor drops in k(cat) and K(M), but the catalytic efficiency (k(cat)/K(M)) was practically unaffected. Temperature-induced unfolding-refolding of both TIM samples displayed hysteresis cycles, indicative of processes far from equilibrium. Kinetic studies showed that the rate constant for unfolding was about three-fold larger in the mutant than in wild-type TIM. However, more drastic changes were found in the kinetics of refolding: upon mutation, the rate-limiting step changed from a second-order (at submicromolar concentrations) to a first-order reaction. These results thus indicate that renaturation of yTIM occurs through a uni-bimolecular mechanism in which refolding of the monomer most likely begins at the C-terminal half of its polypeptide chain. From the temperature dependence of the refolding rate, we determined the change in heat capacity for the formation of the transition state from unfolded monomers. The value for the D225Q mutant, which is about 40% of the corresponding value for yTIM, would implicate the folding of only three quarters of a monomer chain in the transition state.

Effect of a specific inhibitor on the unfolding and refolding kinetics of dimeric triosephosphate isomerase: establishing the dimeric and similarly structured nature of the main transition states on the forward and backward reactions.

2-Phosphoglycolate (PGA), a strong competitive inhibitor of the dimeric enzyme triosephosphate isomerase (TIM), brings about a large decrease in the unfolding rate constant of the protein. The data set of rate constants versus ligand concentration may be equally well explained by regarding either a monomeric or a dimeric transition state (TS). However, if the thermodynamics for binding of PGA to native TIM is taken into account, it becomes clear that a dimeric TS is the right assumption. Furthermore, by studying the effect of the ligand on the second-order refolding reaction, we found results indicating similar PGA-binding affinities to be present in the transition states for the rate-limiting steps of the forward and backward reactions. Most likely, therefore, both TS resemble each other in respect to the active site architecture. It should be mentioned, however, that our data do not rule out the possible occurrence of an unstable, (partially) folded monomeric intermediate, which would rapidly interconvert with the unfolded monomer.

Conserved cysteine 126 in triosephosphate isomerase is required not for enzymatic activity but for proper folding and stability.

In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in the folding of wtTIM, as indicated by spectroscopic studies. C126S and C126A have K(M) and k(cat) values that are concomitantly reduced by only 4-fold and 1.5-fold, respectively, compared to those of wtTIM; in either case, however, the catalytic efficiency (k(cat)/K(M)) of the enzyme is barely affected. The affinity of mutated TIMs for the competitive inhibitor 2-phosphoglycolate augmented also slightly. In contrast, greater susceptibility to thermal denaturation resulted from mutation of Cys126, especially when it was changed to Ser. By using values of the rate constants for unfolding and refolding, we estimated that, at 25 degrees C, C126A and C126S are less stable than wtTIM by about 5.0 and 9.0 kcal mol(-)(1), respectively. Moreover, either of these mutations slows down the folding rate by a factor of 10 and decreases the recovery of the active enzyme after thermal unfolding. Thus, Cys126 is required for proper stability and efficient folding of TIM rather than for enzymatic catalysis.