RESUMO
The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications.
Assuntos
Núcleosídeo-Difosfato Quinase/química , Nucleotídeos de Purina/química , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Adenilil Imidodifosfato/química , Animais , Sítios de Ligação , Varredura Diferencial de Calorimetria/métodos , Cristalografia por Raios X , Dictyostelium/enzimologia , Guanosina Difosfato/química , Humanos , Inosina Difosfato/química , Myxococcus xanthus/enzimologia , Termodinâmica , Nucleotídeos de Timina/química , TitulometriaRESUMO
The reaction of copper-free lentil seedlings amine oxidase with substrates has been studied. While devoid of catalytic activity, this enzyme preparation is still able to oxidize two moles of substrate and to release two moles of aldehyde and two moles of ammonia per mole of dimeric protein. The same stoichiometry has been determined on the native enzyme in the absence of oxygen. Although copper is essential for the reoxidation of the reduced enzyme, a binding of oxygen to the copper-free protein has been demonstrated.
Assuntos
Amina Oxidase (contendo Cobre) , Fabaceae/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Plantas Medicinais , Anaerobiose , Cobre/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Sementes/enzimologiaRESUMO
The reaction of copper amine oxidases from beef plasma and lentil seedlings with hydrazine derivatives has been studied. A 1:1 stoichiometry was always found for the irreversible binding to the dimeric proteins. The formation of the adduct does not require the presence of oxygen or copper. Substrates compete with hydrazine derivatives for the binding to the enzymes. The binding of hydrazines and of substrate has different effects on the EPR spectra of enzymic copper.