Estudos cineticos, fisico-quimicos e conformacional da fosfatase acida de sementes de mamona (Ricinus communis)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2001

RESUMO

This work contains results about kinetic, physico-chemical and conformationalstudies of castor bean seed acid phosphatase. About kinetic approaches we studied the effect of inhibitors and determined the amino acids present in the active site. Inorganic phosphate, molybidate and o vanadate were competitive inhibitors, with Ki values of 0,14 mM, 10 x 10-6 mM e 1,6 X 10-3 mM, respectively. On the other hand, fluoride inhibition was of the mixed type, with Kic and Kiu values of 0.91 mM and 0.42 mM, respectively. The thermal stability studies showed that the enzyme lost 80% of activity when incubated at 60°C for 15 mino But, in the presence of 10 mM of p-nitrophenol and Pi the enzyme lost 40% of activity. 5tudying the amino acids present in the active site by using modifying reagents, we observed that castor bean seed acid phosphatase was inactivated 50% in the presence of IAA, that acted sulfhydryl groups, in an inactivation time- and concentration-dependent. The acid phosphatase has two cysteines in its active site, with bimolecular constant value of 2.9 X 10-4 M-1 S-I. In the physico-chemical and conformational approaches, we determined the isoeletric point, the carbohydrate composition and denaturation studies in the presence of caotropic agents and temperature. The acid phosphatase has a pI value of 4.6 and contained 40% of carbohydrate in its structure, and glucose was the only monossacharide found in the composition. The enzyme denaturation in the presence of caotropic agents, as urea and GndHCl, showed to be reversible. The thermal parameters such as f1G (HzO), DlIz and m were determined to be 4.9 Kcal mol-l, 4.95 M and 0.991 Kcal mol-l, in the presence of urea, and 4.98 Kcal mol-l, 2.35 M and 2.118 Kcal mol-l, in the presence of GdnHCl, respectively. In the thermal denaturation, the enzyme unfolded as aggregate when the temperature was ligher than 90°C. This aggregation was not observed when the enzyme was incubated in the presence of 10 mM pNP and Pi. In both cases the process of denaturation was irreversible. Finally, we studied some properties of the enzyme after carbohydrate remotion by endoglicosidase H. We observed an additional optimum pH of 3.5, a decrease in the specificity constant using pNPP as substrate, a increasing in the pI value and a decrease in the carbohydrate heterogeneity migration in SDS-PAGE. This work contains also some germination studies of castor bean seeds, with determination of acid phosphates activity using pNPP, Tyr-P and PPi as substrates. The peaks of acid phosphatase activity using pNPP, PEP, PPi and Tyr-P as substrates were 7th, 5th and 9th, respectively. The protein concentration increase during germination whi le phosphate concentration decrease

ASSUNTO(S)

enzimas mamona fosfatase acida

ACESSO AO ARTIGO

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