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The reasons underlying the oligomeric nature of some proteins such as triosephosphate isomerase (TIM) are unclear. It has been proposed that this enzyme is an oligomer, mainly because of its stability rather than for functional reasons. To address this issue, the reversible denaturation and renaturation of the homodimeric TIM from baker's yeast ( Saccharomyces cerevisiae ) induced by guanidinium chloride and urea have been characterized by spectroscopic, functional and hydrodynamic techniques. The unfolding and refolding of this enzyme are not coincident after 'conventional' equilibrium times. Unfolding experiments did not reach equilibrium, owing to a very slow dissociation and/or unfolding process. By contrast, equilibrium was reached in the refolding direction. The simplest equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer. The Gibbs energy changes for monomer folding (delta G (0)(fold) = -16.6+/-0.7 kJ x mol(-1)) and monomer association (delta G (0)(assoc) = -70.3+/-1.1 kJ x mol(-1)) were calculated from data obtained in the two denaturants. From an analysis of the present data and data from the literature on the stability of TIM from different species and for other beta/alpha barrels, and model simulations on the effect of stability in the catalytic activity of the enzyme, it is concluded that the low stability of the monomers is neither the only, nor the main, cause for the dimeric nature of TIM. There is interplay between function and stability.

Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.

Methicillin-resistant Staphylococcus auerus (MRSA) strains are having a major impact worldwide, and due to their resistance to all β-lactams, an urgent need for new drugs is emerging. In this regard, the shikimate pathway is considered to be one of the metabolic features of bacteria and is absent in humans. Therefore enzymes involved in this route, such as shikimate dehydrogenase (SDH), are considered excellent targets for discovery of novel antibacterial drugs. In this study, the SDH from MRSA (SaSDH) was characterized. The results showed that the enzyme is a monomer with a molecular weight of 29 kDa, an optimum temperature of 65 °C, and a maximal pH range of 9-11 for its activity. Kinetic studies revealed that SDH showed Michaelis-Menten kinetics toward both substrates (shikimate and NADP + ). Initial velocity analysis suggested that SaSDH catalysis followed a sequential random mechanism. Additionally, a tridimensional model of SaSDH was obtained by homology modeling and validated. Through virtual screening three OPEN ACCESSMolecules 2014, 19 4492 inhibitors of SaSDH were found (compounds 238, 766 and 894) and their inhibition constants and mechanism were obtained. Flexible docking studies revealed that these molecules make interactions with catalytic residues. The data of this study could serve as starting point in the search of new chemotherapeutic agents against MRSA.

Polyphenol oxidase (PPO) is a critical molecule because of its participation in enzymatic browning in fruits and vegetables during ripening, handling, storage, and processing. In this contribution, PPO was extracted, purified, and characterized from Golden Delicious apples, PPO activity was increased 319‐fold with a yield of 1.6%. A monomer of 58 kDa was obtained by gel filtration and SDS‐PAGE; the optimum temperature and pH were 35°C and 6.0, respectively; the best catalytic efficiencies (Vmax/Km ratios [U/(mg·mM)]) were 15,402 for 4‐methylcatechol and 5,941 for pyrogallol. The most efficient inhibitors were sodium metabisulfite (≥1 mM) and tropolone (≥10 mM). PPO activity decreased as the temperature increased and thermal inactivation was achieved at 80°C in 5 min. An Ea of 69.2 kJ/mol was estimated from the kinetic inactivation at different temperatures. Microwave irradiation inactivates PPO after 120 s in crude extract and after 60 s in fresh apple juice. Practical applications According to FAO (Food and Agricultural Organization) data, Mexico produced about 858,608 tons of apple in 2013, Mexico is in 30th place in the world overall apple production. It is important to characterize the Polyphenol Oxidase (PPO) from Golden Delicious apple and provide data on its inactivation. PPO has been considered a crucial molecule because of its participation in enzymatic browning in several fruit and vegetables during ripening, handling, storage, and processing, being responsible for economical loses for producers and industry. In this contribution PPO was extracted, purified, and partially characterized from Mexican Golden Delicious apples by fast protein liquid chromatography. Also some inactivation methods like the use of several inhibitors and antibrowning agents, thermal and microwave inactivation where tested. The information obtained in this research is important for better understanding of PPO from Golden Delicious apple and gives information on ways to optimize inactivation process of the purified enzyme or in apple juice.