Solvent and thermal stability, and pH kinetics, of proline-specific dipeptidyl peptidase IV-like enzyme from bovine serum

Proline-specific dipeptidyl peptidase-like (DPP IV; EC 3.4.14.5) activity in bovine serum has attracted little attention despite its ready availability and the paucity of useful proline-cleaving enzymes. Bovine serum DPP IV-like peptidase is very tolerant of organic solvents, particularly acetonitrile: upon incubation for 1 h at room temperature in 70% acetonitrile, 47% dimethylformamide, 54% DMSO and 33% tetrahydrofuran (v/v concentrations) followed by dilution into the standard assay mixture, the enzyme retained half of its aqueous activity. As for thermal performance in aqueous buffer, its relative activity increased up to 50 ◦C. Upon thermoinactivation at 71 ◦C, pH 8.0 (samples removed periodically, cooled on ice, then assayed under optimal conditions), residual activities over short times fit a first-order decay with a k-value of 0.071±0.0034 min−1. Over longer times, residual activities fit to a double exponential decay with k1 and k2 values of 0.218±0.025 min−1 (46±4% of overall decay) and 0.040±0.002 min−1 (54±4% of overall decay), respectively. The enzyme’s solvent and thermal tolerances suggest that it may have potential for use as a biocatalyst in industry. Kinetic analysis with the fluorogenic substrate Gly-Pro-7-aminomethylcoumarin over a range of pH values indicated two pK values at 6.18±0.07 and at 9.70±0.50. We ascribe the lower value to the active site histidine; the higher may be due to the active site serine or to a free amino group in the substrate

Physarum nitric oxide synthases: genomic structures and enzymology of recombinant proteins

Physarum polycephalum expresses two closely related, calcium-independent NOSs (nitric oxide synthases). In our previous work, we showed that both NOSs are induced during starvation and apparently play a functional role in sporulation. In the present study, we characterized the genomic structures of both Physarum NOSs, expressed both enzymes recombinantly in bacteria and characterized their biochemical properties. Whereas the overall genomic organization of Physarum NOS genes is comparable with various animal NOSs, none of the exon–intron boundaries are conserved. Recombinant expression of clones with various N-termini identified N-terminal amino acids essential for enzyme activity, but not required for haem binding or dimerization, and suggests the usage of non-AUG start codons for Physarum NOSs. Biochemical characterization of the two Physarum isoenzymes revealed different affinities for L-arginine, FMN and 6R-5,6,7,8-tetrahydro-L-biopterin

Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells

ABSTRACT A recombinant adenovirus containing the cDNA of human neuronal nitric-oxide synthase (nNOS) was constructed to characterize the interaction of nNOS with N- [(1,3-benzodioxol-5-yl

Exploring the Role of CYP3A4 Mediated Drug Metabolism in the Pharmacological Modulation of Nitric Oxide Production

Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation, and cytochrome P450, the major enzymes involved in drug metabolism, share striking similarities. Therefore, it makes sense that cytochrome P450 drug mediated biotransformations might play an important role in the pharmacological modulation of nitric oxide synthase. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and nitric oxide modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes in order assess the implication of CYP450 activities over production of nitric oxide. In vitro systems based in human liver microsomes and activated mouse macrophages were developed for these purposes. Additionally in vitro production the hepatic metabolites of dual inhibitor, roxithromycin, was investigated achieving the identification and isolation of main hepatic biotransformation products. Our results suggested that for some macrolide compounds, the cytochrome P450 3A4 derived drug metabolites have an important effect on nitric oxide production and might critically contribute to the pharmacological immunomodulatory activity observed.This work was supported by the Spanish Ministry of Economy and Competitivity (SAF2011-29648) and Junta de Andalucía (AGR-6826 and CTS 164) with funds from the European Union; FA is a predoctoral fellow of Junta de Andalucia; MR is a postdoctoral fellow of CIBER-EHD. The CIBEREHD is funded by the Instituto de Salud Carlos III. The MEDINA authors disclosed the receipt of financial support from Fundación MEDINA, a public-private partnership of Merck Sharp and Dohme de España S.A./Universidad de Granada/Junta de Andalucía. In the case of Noemi Vergara Segura, she was a CEIBioTic fellow from the program at the Granada University. The results presented in this work will be compiled in the doctoral thesis (knowledge area code 32089 and subject code 320903) entitled “Evaluación de la actividad de los metabolitos hepáticos derivados de compuestos inmunomodulares e inhibidores de GSK3,” carried out currently by JP at FUNDACIÓN MEDINA in collaboration with the department of Pharmacology at the University of Granada, being the thesis directors, FV (FUNDACIÓN MEDINA) and JG (Universidad de Granada)

Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding

Constitutive isoforms of nitric oxide synthase (NOS) are activated by transient binding of Ca(2+)/Calmodulin. Here, we characterize the binding of Calmodulin to purified neuronal NOS (nNOS). [125I]Calmodulin bound to a single class of non-interacting and high affinity sites on nNOS. [125I]Calmodulin binding achieved rapid saturation, was linear with nNOS concentration, and exhibited a strict dependence on [Ca(2+)]. Neither affinity nor extent of [125I]Calmodulin binding was affected by L-arginine, NADPH or Tetrahydrobiopterin. Native Calmodulin and engineered Calmodulin homologs [i.e., duplicated N-terminal (CaMNN)] potently displaced [125I]Calmodulin. CaMNN supported nNOS catalysis, but required approximately five-fold more Ca(2+) for comparable activity with native Calmodulin. Taken with results from kinetic analyses of [125I]Calmodulin association and dissociation, our findings suggest four sequential steps in activation of nNOS by Calmodulin: (1) Ca(2+) binds to Calmodulin's C-lobe, (2) the C-lobe of Calmodulin binds NOS, (3) Ca(2+) binds to the N-lobe of Calmodulin, and (4) the N-lobe binds to nNOS. Activation of nNOS only occurs after completion of step (4), with the displacement of nNOS's autoinhibitory insert. Upon intracellular Ca(2+) sequestration, deactivation of nNOS would proceed in reverse order

Thermodynamic Characterization of Five Key Kinetic Parameters That Define Neuronal Nitric Oxide Synthase Catalysis

NO synthase (NOS) enzymes convert L-arginine to NO in two sequential reactions whose rates (kcat1 and kcat2) are both limited by the rate of ferric heme reduction (kr). An enzyme ferric heme –NO complex forms as an immediate product complex and then undergoes either dissociation (at a rate that we denote as kd) to release NO in a productive manner, or reduction (kr) to form a ferrous heme –NO complex that must react with O2 (at a rate that we denote as kox) in a NO dioxygenase reaction that regenerates the ferric enzyme. The interplay of these five kinetic parameters (kcat1, kcat2, kr, Kd and kox) determines NOS specific activity, O2 concentration response, and pulsatile versus steady-state NO generation. In the present study, we utilized stopped-flow spectroscopy and single catalytic turnover methods to characterize the individual temperature dependencies of the five kinetic parameters of rat neuronal NOS. We then incorporated the measured kinetic values into computer simulations of the neuronal NOS reaction using a global kinetic model to comprehensively model its temperature dependent catalytic behaviours. The results obtained provide new mechanistic insights and also reveal that the different temperature dependencies of the five kinetic parameters significantly alter neuronal NOS catalytic behaviours and NO release efficiency as a function of temperature