Navegando por Palavras-chave "Michaelis-menten kinetics"

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    Caracterização bioquímica da enzima antranilato fosforibosil transferase de cryptococcus neoformans
    (Universidade Federal de São Paulo (UNIFESP), 2015-09-12) Chagas, Rayane Arraes Jardim [UNIFESP]; Alfonso, Martin Rodrigo Alejandro Wurtele [UNIFESP]; http://lattes.cnpq.br/0625673643875019; Universidade Federal de São Paulo (UNIFESP)
    The number of patients with opportunist fungal diseases, e.g. criptococosis, has increased in recent years due to the higher occurrence of immunosuppressed patients. This phenomenon can be associated, for example, to the appearance of AIDS, to the utilization of new medical practices, like immunosuppressive therapies, and to the indiscriminate use of antimicrobial drugs. Nevertheless, the number of available antifungal drugs has remained sparse, mainly due to the low availability of new targets with highly selective toxicity, since the fungal cells are similar to the human ones, both eukaryotes. The tryptophan biosynthetic pathway is absent in human beings and past studies showed a decreasing virulence in microorganisms in which mutations caused the absence of the production of this amino acid. Thus, the study of this biosynthetic pathway is important to develop new drugs with high selective toxicity against fungal diseases. The tryptophan biosynthetic pathway is composed of five enzymes, which are: anthranilate synthase, anthranilate phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, indole-3-glycerol phosphate synthase, and tryptophan synthase. In the human-pathogen model Cryptococcus neoformans, the gene APRT codifies the anthranilate phosphoribosyltransferase enzyme, which, in turn, catalyzes the second step of this pathway, catalyzing the transfer of the phosphoribosyl group of phosphoribosyl pyrophosphate (PRPP) to anthranilate , thus forming phosphoribosyl anthranilate. Therefore, the aim of this work was to clone, to express, and to purify in a recombinant form this pathogenic organism?s enzyme and to analyze its activity through fluorescence assay. Activity of the enzyme was verified by a Michaelis-Menten kinetic. Hence, it was possible to obtain the following constants: Km = 1,3995 ?M ± 0,6 ?M for anthranilate, Km = 0,3745 mM ± 0,14 mM for PRPP, and kcat = 0,0037s-1 ± 0,0005s-1. Biochemical characterization of this enzyme will enable future chemical compounds screens to find specific inhibitors. This, in turn, will allow the development of new techniques against this opportunist pathogen, thus demonstrating the expected impact of this applied research area.