Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Enzymatic Logic Gates with Noise-Reducing Sigmoid Response

Biochemical computing is an emerging field of unconventional computing that attempts to process information with biomolecules and biological objects using digital logic. In this work we survey filtering in general, in biochemical computing, and summarize the experimental realization of an and logic gate with sigmoid response in one of the inputs. The logic gate is realized with electrode-immobilized glucose-6-phosphate dehydrogenase enzyme that catalyzes a reaction corresponding to the Boolean and functions. A kinetic model is also developed and used to evaluate the extent to which the performance of the experimentally realized logic gate is close to optimal.

Bayesian L-optimal exact design of experiments for biological kinetic models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinetic properties of glycerophosphate oxidase isolated from dry baker's yeast

The glycerophosphate oxidase is a flavoprotein responsible for the catalysis of the oxidation of the glycerophosphate to dihydroxyacetone phosphate, through the reduction of the oxygen to hydrogen peroxide. The glycerophosphate oxidase from baker's yeast was specific for L-alpha-glycerol phosphate. It was estimated by monitoring the consumption of oxygen with an oxygraph. An increase of 32% in consumption of oxygen was obtained when the enzyme was concentrated 16-fold. The assay of enzyme was determined by the peroxidase chromogen method followed at 500 nm. The procedure for the standardization of the activity of the glycerophosphate oxidase from baker's yeast was accomplished, and the pH and temperature stability showed that the enzyme presented a high stability at pH 8.0, and the thermal stability was maintained up to 60 degrees C during I h. Such method allowed quantifying in the range 92-230 mM of glycerol phosphate, an important intermediate metabolite from lipid biosynthesis and glycolytic routes. (C) 2007 Elsevier B.V. All rights reserved.

A comparative study of protein and enzymatic activity in venoms of some common wasps (Hymenoptera: Vespidae) from São Paulo State

The Hymenoptera Aculeata venoms, with few exceptions, have been poorly studied and characterized. Nevertheless, they have raised increasing interest due to their medical importance, since accidents with these insects are fairly frequent in Brazil and may cause severe allergic reactions. The objectives of the present work were the quantitative characterization of the main allergenic enzymes present in the venom of the species Polybia paulista, Polybia ignobilis, Polistes simillimus, and Agelaia pallipes pallipes through biochemical assays for the determination of total protein content, as well as the level of the enzymatic activity of phospholipase, hyaluronidase, acid phosphatase and esterase. These results, in addition to providing biochemical knowledge about the venom of the species in question, also supply studies that allow phylogenetic inferences among them.

Antioxidant properties of brazilian tropical fruits by correlation between different assays

Four different assays (the Folin-Ciocalteu, DPPH, enzymatic method, and inhibitory activity on lipid peroxidation) based on radically different physicochemical principles and normally used to determine the antioxidant activity of food have been confronted and utilized to investigate the antioxidant activity of fruits originated from Brazil, with particular attention to more exotic and less-studied species (jurubeba, Solanum paniculatum; pequi, Caryocar brasiliense; pitaya, Hylocereus undatus; siriguela, Spondias purpurea; umbu, Spondias tuberosa) in order to (i) verify the correlations between results obtained by the different assays, with the final purpose to obtain more reliable results avoiding possible measuring-method linked mistakes and (ii) individuate the more active fruit species. As expected, the different methods give different responses, depending on the specific assay reaction. Anyhow all results indicate high antioxidant properties for siriguela and jurubeba and poor values for pitaya, umbu, and pequi. Considering that no marked difference of ascorbic acid content has been detected among the different fruits, experimental data suggest that antioxidant activities of the investigated Brazilian fruits are poorly correlated with this molecule, principally depending on their total polyphenolic content. © 2013 Elena Gregoris et al.

Kinetic role of a histidine residue in the T1 copper site of the laccase from Rigidoporus lignosus

Laccases (benzendiol:oxygen oxidoreductases; EC 1.10.3.2) catalyze the oxidation of a broad range of substrates, such as polyphenols, dyes and pollutants, and thus these enzymes are widely applied in industrial, biotechnological and environmental fields. In order to improve their biotechnological applications, a deep knowledge of structural factors involved in controlling their activity, in various experimental conditions and on different substrates, is required. In the present study, a laccase from the mushroom Rigidoporus lignosus was kinetically characterized. In particular, the stability, the effects of pH, ionic strength and fluoride ion concentration on the kinetic parameters were investigated, using three di-hydroxy-benzene isomers (1,2-dihydroxy-benzene, 1,3-dihydroxy-benzene and 1,4-dihydroxy-benzene) as substrates. The catalytic constant values of the laccase showed a bell-shaped pH profile, with the same optimum pH and pK(a) values for all tested substrates. This behavior appears to be due to the presence of an ionizable residue in the enzyme active site. To identify this residue, the enzyme was derivatized with diethylpyrocarbonate to modify accessible histidine residues, which, according to structural data, are present in the active site of this enzyme. The kinetic behavior of the derivatized laccase was compared with that of the native enzyme and the derivatized residues were identified by mass spectrometry. Mass spectrometry and kinetic results suggest the main role of His-457 in the control of the catalytic activity of laccase from R. lignosus. (C) 2013 Elsevier B.V. All rights reserved.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: Insights into substrate selectivity and kinetic behavior

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synthesis of Structured Lipids by Enzymatic Interesterification of Milkfat and Soybean Oil in a Basket-Type Stirred Tank Reactor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural and kinetic studies of Schistosoma mansoni adenylate kinases

The human parasite Schistosoma mansoni is totally dependent on the purine salvage pathway in order to supply large quantities of purine precursors for its energy and DNA biosynthetic needs. Adenylate kinase (ADK) is responsible for the conversion of AMP (produced by the adenosine kinase reaction) into ADP, which is subsequently converted into ATP by nucleoside diphosphate kinase (NDPK). ADK and NDPK are the most active enzymes of the pathway, probably reflecting an evolutionary adaptation due to the intense use of the branch in which they participate. However, notwithstanding their importance very little information has been accumulated found regarding these enzymes. In this work two adenylate kinases from S. mansoni were cloned and heterologously expressed in Escherichia coil. The purified products were utilized in activity assays, and displayed kinetic parameters similar to the corresponding human orthologous proteins. The cytosolic S. mansoni ADK was crystallized and its structure solved allowing us to detect a difference in the nucleotide binding site when compared with the human ortholog. (C) 2012 Elsevier B.V. All rights reserved.

Substrate specificity of the Chamaerops excelsa palm tree peroxidase. A steady-state kinetic study

The steady state kinetic mechanism of the H(2)O(2)-supported oxidation of different organic substrates by peroxidase from leaves of Chamaerops excelsa palm trees (CEP) has been investigated. An analysis of the initial rates vs. H(2)O(2) and reducing substrate concentrations is consistent with a substrate-inhibited Ping-Pong Bi Bi reaction mechanism. The phenomenological approach expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and leads to an interpretation of the effects in terms of the kinetic parameters K(m)(H2O2)center dot K(m)(AH2)center dot k(cat)center dot K(SI)(AH2) and of the microscopic rate constants k(1) and k(3) of the shared three-step catalytic cycle of peroxidases. (C) 2011 Elsevier B.V. All rights reserved.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

Biochemical analysis of the phosphatase domain of the human soluble epoxide hydrolase (sEH)

Die lösliche Epoxidhydrolase (sEH) gehört zur Familie der Epoxidhydrolase-Enzyme. Die Rolle der sEH besteht klassischerweise in der Detoxifikation, durch Umwandlung potenziell schädlicher Epoxide in deren unschädliche Diol-Form. Hauptsächlich setzt die sEH endogene, der Arachidonsäure verwandte Signalmoleküle, wie beispielsweise die Epoxyeicosatrienoic acid, zu den entsprechenden Diolen um. Daher könnte die sEH als ein Zielenzym in der Therapie von Bluthochdruck und Entzündungen sowie diverser anderer Erkrankungen eingesetzt werden. rnDie sEH ist ein Homodimer, in dem jede Untereinheit aus zwei Domänen aufgebaut ist. Das katalytische Zentrum der Epoxidhydrolaseaktivität befindet sich in der 35 kD großen C-terminalen Domäne. Dieser Bereich der sEH s wurde bereits im Detail untersucht und nahezu alle katalytischen Eigenschaften des Enzyms sowie deren dazugehörige Funktionen sind in Zusammenhang mit dieser Domäne bekannt. Im Gegensatz dazu ist über die 25 kD große N-terminale Domäne wenig bekannt. Die N-terminale Domäne der sEH wird zur Haloacid Dehalogenase (HAD) Superfamilie von Hydrolasen gezählt, jedoch war die Funktion dieses N-terminal Domäne lange ungeklärt. Wir haben in unserer Arbeitsgruppe zum ersten Mal zeigen können, dass die sEH in Säugern ein bifunktionelles Enzym ist, welches zusätzlich zur allgemein bekannten Enzymaktivität im C-terminalen Bereich eine weitere enzymatische Funktion mit Mg2+-abhängiger Phosphataseaktivität in der N-terminalen Domäne aufweist. Aufgrund der Homologie der N-terminalen Domäne mit anderen Enzymen der HAD Familie wird für die Ausübung der Phosphatasefunktion (Dephosphorylierung) eine Reaktion in zwei Schritten angenommen.rnUm den katalytischen Mechanismus der Dephosphorylierung weiter aufzuklären, wurden biochemische Analysen der humanen sEH Phosphatase durch Generierung von Mutationen im aktiven Zentrum mittels ortsspezifischer Mutagenese durchgeführt. Hiermit sollten die an der katalytischen Aktivität beteiligten Aminosäurereste im aktiven Zentrum identifiziert und deren Rolle bei der Dephosphorylierung spezifiziert werden. rnrnAuf Basis der strukturellen und möglichen funktionellen Ähnlichkeiten der sEH und anderen Mitgliedern der HAD Superfamilie wurden Aminosäuren (konservierte und teilweise konservierte Aminosäuren) im aktiven Zentrum der sEH Phosphatase-Domäne als Kandidaten ausgewählt.rnVon den Phosphatase-Domäne bildenden Aminosäuren wurden acht ausgewählt (Asp9 (D9), Asp11 (D11), Thr123 (T123), Asn124 (N124), Lys160 (K160), Asp184 (D184), Asp185 (D185), Asn189 (N189)), die mittels ortsspezifischer Mutagenese durch nicht funktionelle Aminosäuren ausgetauscht werden sollten. Dazu wurde jede der ausgewählten Aminosäuren durch mindestens zwei alternative Aminosäuren ersetzt: entweder durch Alanin oder durch eine Aminosäure ähnlich der im Wildtyp-Enzym. Insgesamt wurden 18 verschiedene rekombinante Klone generiert, die für eine mutante sEH Phosphatase Domäne kodieren, in dem lediglich eine Aminosäure gegenüber dem Wildtyp-Enzym ersetzt wurde. Die 18 Mutanten sowie das Wildtyp (Sequenz der N-terminalen Domäne ohne Mutation) wurden in einem Expressionsvektor in E.coli kloniert und die Nukleotidsequenz durch Restriktionsverdau sowie Sequenzierung bestätigt. Die so generierte N-terminale Domäne der sEH (25kD Untereinheit) wurde dann mittels Metallaffinitätschromatographie erfolgreich aufgereinigt und auf Phosphataseaktivität gegenüber des allgemeinen Substrats 4-Nitophenylphosphat getestet. Diejenigen Mutanten, die Phosphataseaktivität zeigten, wurden anschließend kinetischen Tests unterzogen. Basiered auf den Ergebnissen dieser Untersuchungen wurden kinetische Parameter mittels vier gut etablierter Methoden berechnet und die Ergebnisse mit der „direct linear blot“ Methode interpretiert. rnDie Ergebnisse zeigten, dass die meisten der 18 generierten Mutanten inaktiv waren oder einen Großteil der Enzymaktivität (Vmax) gegenüber dem Wildtyp verloren (WT: Vmax=77.34 nmol-1 mg-1 min). Dieser Verlust an Enzymaktivität ließ sich nicht durch einen Verlust an struktureller Integrität erklären, da der Wildtyp und die mutanten Proteine in der Chromatographie das gleiche Verhalten zeigten. Alle Aminosäureaustausche Asp9 (D9), Lys160 (K160), Asp184 (D184) und Asn189 (N189) führten zum kompletten Verlust der Phosphataseaktivität, was auf deren katalytische Funktion im N-terminalen Bereich der sEH hindeutet. Bei einem Teil der Aminosäureaustausche die für Asp11 (D11), Thr123 (T123), Asn124 (N124) und Asn185 (D185) durchgeführt wurden, kam es, verglichen mit dem Wildtyp, zu einer starken Reduktion der Phosphataseaktivität, die aber dennoch für die einzelnen Proteinmutanten in unterschiedlichem Ausmaß zu messen war (2 -10% and 40% of the WT enzyme activity). Zudem zeigten die Mutanten dieser Gruppe veränderte kinetische Eigenschaften (Vmax allein oder Vmax und Km). Dabei war die kinetische Analyse des Mutanten Asp11  Asn aufgrund der nur bei dieser Mutanten detektierbaren starken Vmax Reduktion (8.1 nmol-1 mg-1 min) und einer signifikanten Reduktion der Km (Asp11: Km=0.54 mM, WT: Km=1.3 mM), von besonderem Interesse und impliziert eine Rolle von Asp11 (D11) im zweiten Schritt der Hydrolyse des katalytischen Zyklus.rnZusammenfassend zeigen die Ergebnisse, dass alle in dieser Arbeit untersuchten Aminosäuren für die Phosphataseaktivität der sEH nötig sind und das aktive Zentrum der sEH Phosphatase im N-terminalen Bereich des Enzyms bilden. Weiterhin tragen diese Ergebnisse zur Aufklärung der potenziellen Rolle der untersuchten Aminosäuren bei und unterstützen die Hypothese, dass die Dephosphorylierungsreaktion in zwei Schritten abläuft. Somit ist ein kombinierter Reaktionsmechanismus, ähnlich denen anderer Enzyme der HAD Familie, für die Ausübung der Dephosphorylierungsfunktion denkbar. Diese Annahme wird gestützt durch die 3D-Struktur der N-terminalen Domäne, den Ergebnissen dieser Arbeit sowie Resultaten weiterer biochemischer Analysen. Der zweistufige Mechanismus der Dephosphorylierung beinhaltet einen nukleophilen Angriff des Substratphosphors durch das Nukleophil Asp9 (D9) des aktiven Zentrums unter Bildung eines Acylphosphat-Enzym-Zwischenprodukts, gefolgt von der anschließenden Freisetzung des dephosphorylierten Substrats. Im zweiten Schritt erfolgt die Hydrolyse des Enzym-Phosphat-Zwischenprodukts unterstützt durch Asp11 (D11), und die Freisetzung der Phosphatgruppe findet statt. Die anderen untersuchten Aminosäuren sind an der Bindung von Mg 2+ und/oder Substrat beteiligt. rnMit Hilfe dieser Arbeit konnte der katalytischen Mechanismus der sEH Phosphatase weiter aufgeklärt werden und wichtige noch zu untersuchende Fragestellungen, wie die physiologische Rolle der sEH Phosphatase, deren endogene physiologische Substrate und der genaue Funktionsmechanismus als bifunktionelles Enzym (die Kommunikation der zwei katalytischen Einheiten des Enzyms) wurden aufgezeigt und diskutiert.rn