Structural insights into the molecular basis responsible for the effects of immobilization on the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi and human

Journal of the Brazilian Chemical Society

Rafael V. C. Guido²  Carmen L. Cardoso¹  Marcela C. De Moraes¹  Adriano D. Andricopulo²  Quezia B. Cass¹  Glaucius Oliva² 

[1] ,Universidade de São Paulo Instituto de Física de São Carlos Centro de Biotecnologia Molecular EstruturalSão Carlos SP ,Brazil

关键词: immobilized enzymes reactors;    enzymes;    kinetic parameters;    structural analysis;    drug design;   

DOI  :  10.1590/S0103-50532010001000008

来源: SciELO

【 摘 要 】

The development of fast and reliable methods for the identification of new bioactive compounds is of utmost importance to boost the process of drug discovery and development. Immobilized enzyme reactors (IMERs), integrated with high performance liquid chromatography (HPLC), are attractive and versatile tools for screening collections consisting of natural products and synthetic small molecules. Standard kinetic parameters of the immobilized enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from both Trypanosoma cruzi de and human have been determined (T. cruzi: K M G3P = 0.50 mmol L-1; K M NAD+ = 0.67 mmol L-1; humana: K M G3P = 3.7 mmol L-1; K M NAD+ = 0.75 mmol L-1), and comparisons of these values with those of the parasite and human free enzymes indicate a decrease in the affinity for the immobilized system (T. cruzi: K M G3P = 0.42 mmol L-1; K M NAD+ = 0.26 mmol L-1; humana: K M G3P = 0.16 mmol L-1; K M NAD+ = 0.18 mmol L-1). Interestingly, despite the kinetic differences between the two systems, the immobilized GAPDHs retained the required structural requirements for molecular recognition and biological activity, increasing the stability the enzyme. In the present work, we described an integrated structural analysis which has provided important insights into the molecular basis underlying the effects of immobilization on the ligand-receptor interactions and consequent enzymatic activity and kinetics parameters.

【 授权许可】

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