Kinetic-conceptual model of the hydrolysis of bovine plasma proteins - ALCALASE® 2.9L: The role of inhibition by product.

In the present work, the inhibitory effect of the peptide fractions, obtained through enzymatic hydrolysis of bovine plasma was evaluated, on the enzyme used in the reaction (Alcalase 2.4 L). In this sense, Ultra-filtered peptide fractions of different molecular sizes (A: Fraction>10; B: Fraction 10-3 kDa; and C: Fraction <3 kDa), were used to verify the impact on the total hydrolysis rate. The Fractions between 3 and 10 kDa were refined to fit a conceptual kinetic model which considers inhibition by product and substrate. Additionally, the inactivation of the enzyme through the reaction time was evaluated and its effects incorporated into the model. It was shown that some peptides released in the successive stages of the reaction can in turn inhibit the activity of the hydrolyzing enzyme. The model evaluated suggests a time-varying expression of inhibition parameters as a function of the initial substrate concentration in the reaction. This is based on the kinetic changes of the product profiles for each reaction time in the evaluated operating conditions (S0 variable). A greater inhibitory effect due to the products is evidenced when the reaction occurs with a higher load of the initial substrate (S0 = 20 g/L).

Grandes mujeres científicas: Maud Leonora Menten / Great scientific women: Maud Leonora Menten

Maud Leonora Menten nació en Canadá, tuvo cuatro títulos universitarios: Bachiller en Artes, Master en Fisiología, médica y Doctora en Bioquímica. Trabajó en Estados Unidos, Alemania y Canadá. Trabajó en diferentes áreas: en la distribución de los iones cloruro en el sistema nervioso central, en tumores experimentales y su tratamiento con bromuro de radio, en el equilibrio ácido-base durante la anestesia, en el mecanismo hiperglucemiante de toxinas bacterianas, en el descubrimiento de un mecanismo de acoplamiento en química orgánica y hasta en la electroforesis de las hemoglobinas humanas. Sin embargo, el aporte por el cual es más conocida es su trabajo en el estudio de la cinética enzimática junto a Leonor Michaelis en 1913. El propósito de este trabajo es exponer la vida personal y académica de una científica conocida por la gran mayoría de los profesionales de la salud. La mujer que a principios del siglo XX trabajó con grandes investigadores de Canadá, Estados Unidos y Alemania, cuyos aportes científicos fueron reconocidos muchas décadas después. (AU)

Maud Leonora Menten was born in Canada; she had four university degrees, Bachelor of Arts, Master of Physiology, Physician and Doctor of Biochemistry. She worked in the United States, Germany, and Canada. Maud worked in different areas: the distribution of chloride ions in the central nervous system, experimental tumors and their treatment with radium bromide, the acid-base balance during anesthesia, the hyperglycemic mechanism of bacterial toxins, the discovery of a coupling mechanism in organic chemistry and even the electrophoresis of human hemoglobins. However, the contribution for which she is best known is for her work in the study of enzymatic kinetics with Leonor Michaelis in 1913. The aim of this paper is to expose the personal and academic life of a scientist known to the vast majority of Health professionals. The woman who, at the beginning of the 20th century, worked with great researchers from Canada, the United States and Germany, whose scientific contributions were recognized many decades later. (AU)

Peroxidase as a simultaneous degradation agent of ochratoxin A and zearalenone applied to model solution and beer.

The aim of this study was to evaluate the action of the commercial peroxidase (POD) enzyme (Armoracia rusticana) on the simultaneous degradation of ochratoxin A (OTA) and zearalenone (ZEA) in model solution and beer. For this purpose, the reaction parameters for POD action were optimized, POD application in the degradation of mycotoxins in model solution and beer was evaluated and the kinetic parameters of POD were defined (Michaelis-Menten constant - KM and maximal velocity - Vmax). In the reaction conditions (pH 7, ionic strength of 25 mM, incubation at 30 °C, addition of 26 mM H2O2 and 1 mM potassium ion), POD (0.6 U mL-1) presented the maximum activity for simultaneous degradation of OTA and ZEA of 27.0 and 64.9%, respectively, in model solution after 360 min. The application of POD in beer resulted in the simultaneous degradation of OTA and ZEA of 4.8 and 10.9%, respectively. The kinetic parameters KM and Vmax for degradation of OTA and ZEA were 50 and 10,710 nM and 0.168 and 72 nM min-1, respectively. Therefore, POD can be a promising alternative to mitigate the contamination of OTA and ZEA in model solution and beer, minimizing their effects in humans.

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase.

Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221 , with unit-cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

Zearalenone reduction by commercial peroxidase enzyme and peroxidases from soybean bran and rice bran.

The peroxidase (POD) enzyme, obtained from different sources, has been described in the literature regarding its good results of reduction in concentration or degradation levels of mycotoxins, such as aflatoxin B1, deoxynivalenol and zearalenone (ZEA). This study aimed at evaluating the action of commercial POD and POD from soybean bran (SB) and rice bran (RB) in ZEA reduction in a model solution and the characterisation of the mechanism of enzyme action. POD was extracted from SB and RB in phosphate buffer by orbital agitation. Evaluation of the action of commercial POD and POD from SB and RB in ZEA reduction was carried out in phosphate buffer and aqueous solution, respectively. Parameters of (Michaelis-Menten constant) (KM) and maximal rate (Vmax) were determined in the concentration range from 0.16 to 6 µg mL-1. ZEA reduction was determined and the mechanism of enzyme action was characterised by FTIR and high-pressure liquid chromatography-electrospray tandem mass spectrometry. Commercial POD and POD from RB and SB reduced ZEA concentration by 69.9%, 47.4% and 30.6% in 24 h, respectively. KM values were 39.61 and 8.90 µM, whereas Vmax values were 0.170 and 0.011 µM min-1 for commercial POD and POD from RB, respectively. The characterisation of the mechanism of enzyme action showed the oxidoreductive action of commercial POD in the mycotoxin. The use of commercial POD and POD from agro-industrial by-products, such as SB and RB, could be a promising alternative for ZEA biodegradation.

Species-Specific Inactivation of Triosephosphate Isomerase from Trypanosoma brucei: Kinetic and Molecular Dynamics Studies.

Human African Trypanosomiasis (HAT), a disease that provokes 2184 new cases a year in Sub-Saharan Africa, is caused by Trypanosoma brucei. Current treatments are limited, highly toxic, and parasite strains resistant to them are emerging. Therefore, there is an urgency to find new drugs against HAT. In this context, T. brucei depends on glycolysis as the unique source for ATP supply; therefore, the enzyme triosephosphate isomerase (TIM) is an attractive target for drug design. In the present work, three new benzimidazole derivatives were found as TbTIM inactivators (compounds 1, 2 and 3) with an I50 value of 84, 82 and 73 µM, respectively. Kinetic analyses indicated that the three molecules were selective when tested against human TIM (HsTIM) activity. Additionally, to study their binding mode in TbTIM, we performed a 100 ns molecular dynamics simulation of TbTIM-inactivator complexes. Simulations showed that the binding of compounds disturbs the structure of the protein, affecting the conformations of important domains such as loop 6 and loop 8. In addition, the physicochemical and drug-like parameters showed by the three compounds suggest a good oral absorption. In conclusion, these molecules will serve as a guide to design more potent inactivators that could be used to obtain new drugs against HAT.

Structural and Enzymatic Characterization of a cAMP-Dependent Diguanylate Cyclase from Pathogenic Leptospira Species.

Leptospira interrogans serovar Copenhageni is a human pathogen that causes leptospirosis, a worldwide zoonosis. The L. interrogans genome codes for a wide array of potential diguanylate cyclase (DGC) enzymes with characteristic GGDEF domains capable of synthesizing the cyclic dinucleotide c-di-GMP, known to regulate transitions between different cellular behavioral states in bacteria. Among such enzymes, LIC13137 (Lcd1), which has an N-terminal cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA (GAF) domain and a C-terminal GGDEF domain, is notable for having close orthologs present only in pathogenic Leptospira species. Although the function and structure of GGDEF and GAF domains have been studied extensively separately, little is known about enzymes with the GAF-GGDEF architecture. In this report, we address the question of how the GAF domain regulates the DGC activity of Lcd1. The full-length Lcd1 and its GAF domain form dimers in solution. The GAF domain binds specifically cAMP (KD of 0.24µM) and has an important role in the regulation of the DGC activity of the GGDEF domain. Lcd1 DGC activity is negligible in the absence of cAMP and is significantly enhanced in its presence (specific activity of 0.13s-1). The crystal structure of the Lcd1 GAF domain in complex with cAMP provides valuable insights toward explaining its specificity for cAMP and pointing to possible mechanisms by which this cyclic nucleotide regulates the assembly of an active DGC enzyme.

In vitro metabolism of the alkaloid piplartine by rat liver microsomes.

Because piplartine (PPT) has demonstrated biological activities, such as cytotoxic, anxiolytic, antidepressant, antifungal and antiplatelet activities, this molecule is a relevant drug candidate. The metabolic fate of drug candidates is an essential requirement in assessing their safety and efficacy. Based on this requirement, the biotransformation of PPT by cytochrome P450 enzymes (CYP) was investigated for the first time. To determine the in vitro enzymatic kinetic parameters, an HPLC method was developed and validated to quantify PPT. All samples were separated on a reversed-phase C18 column using a mobile phase of acetonitrile:water (40:60, v/v). The method exhibited a linear range of 2.4-157.7 µmol/L, with the following calibration curve: y=0.0934 (±0.0010)x+0.0027, r=0.9975. The lower limit of quantitation was verified to be 2.4 µmol/L, with an RSD below 7%. The precision and accuracy were assessed for both within-day and between-day determinations; neither relative standard (RSD%) deviations nor relative errors (RER) exceeded a value of 15%. The mean absolute recovery was 85%, with an RSD value below 6%. The enzymatic kinetic parameters revealed a sigmoidal profile, with V(max)=4.7±0.3 µmol/mg mL⁻¹/min, h=2.5±0.4, S50=44.7±0.3 µmol/L and CL(max)=0.054 µL/min/mg protein, indicating cooperativity behavior. Employing a mammalian model, PPT metabolism yielded two unreported monohydroxylated products (m/z 334). The identification and structural elucidation of the metabolites were performed by comparing their mass spectra with those spectra of the parent drug. For the first time, the in vitro metabolism studies employing microsomes were demonstrated to be a suitable tool for data regarding enzymatic kinetics and for the metabolites formed in the PPT mammalian metabolism.

A non-michaelian behavior of the in vitro metabolism of the pentacyclic triterpene alfa and beta amyrins by employing rat liver microsomes.

Pharmacological studies employing alpha and beta amyrin have demonstrated potential application in several biological activities suggesting their application as promising drugs. In the early drug development, metabolism studies may give important parameters regarding the efficacy and safety of the drug candidate. Therefore, the aim of this work was to determine the enzymatic kinetic parameters of these pentacyclic triterpenes. Chromatographic analyzes were performed using a Shimadzu GC-MS system. The resolution of amyrins was achieved with a DB5-MS column of 0.25 µM film thickness, 30.0 cm length and 0.25 mm diameter. At this condition, the retention times of beta- and alpha-amyrin were 21.3 and 20.2 min, respectively. The proposed method showed to be linear over the concentration range of 0.16-42.18 µM for beta amyrin and 0.11-28.12 µM for alpha amyrin. The lowest concentration quantified by the validated method was 0.16 µM for beta and 0.11 µM for alpha amyrin. The stability study showed that amyrins were stable at room temperature for 12h and at 37°C for 1h. The absolute recovery of the amyrin isomers from the rat microsome was 54.3-59.2%. The enzymatic kinetics presented sigmoidal plots. It was observed a Vmax=0.698 ± 0.022 µmol/mg protein/min, S50=4.4 µM and Hill coefficient of 2.7 ± 0.17 for alpha amyrin and a Vmax=0.775 ± 0.034 µmol/mg protein/min, S50=7.0 µM and Hill coefficient of 2.5 ± 0.21 for beta amyrin. The obtained results give the first clues regarding amyrin metabolism and suggests a more detailed study conducted employing isolated CYP isoforms.