RESUMO
A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 +/- 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca2+, Mg2+, and Mn2+, whereas Cu2+, Zn2+, and Ni2+ ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.
Assuntos
Fabaceae/enzimologia , Plantas Medicinais , Pirofosfatases/isolamento & purificação , Pirofosfatases/metabolismo , Sítios de Ligação , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dietil Pirocarbonato/química , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Fabaceae/crescimento & desenvolvimento , Flavina-Adenina Dinucleotídeo/metabolismo , Concentração de Íons de Hidrogênio , NAD/metabolismo , Pirofosfatases/química , Análise Espectral , Especificidade por SubstratoRESUMO
The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Ki value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.
Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/metabolismo , Inibidores Enzimáticos/farmacologia , Fabaceae/enzimologia , Guanidinas/farmacologia , Rim/enzimologia , Plantas Medicinais , Amilorida/farmacologia , Amina Oxidase (contendo Cobre)/química , Animais , Biguanidas/farmacologia , Ligação Competitiva , Catálise , Inibidores Enzimáticos/farmacocinética , Guanidinas/farmacocinética , Cinética , Espectrofotometria , Relação Estrutura-Atividade , SuínosRESUMO
The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.