Steady-State Kinetics and Mutational Studies of Recombinant Human Thiamin Pyrophosphokinase
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概要
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Thiamin pyrophosphokinase catalyzes the pyrophosphorylation of thiamin to thiamin pyrophosphate in the presence of ATP and Mgtt The kinetic properties of human thiamin pyrophosphokinase (hTPK1) were investigated using purified histidine-tagged recombinant protein. The plots of the initial velocity against MgATP concentrations gave a sigmoidal character when Mg2+/ATP was maintained at 1. However, the addition of an excess amount of Mg2+ resulted in the restoration of activity at lower concentrations of MgATP A steady-state kinetics study led us to conclude that the kinase reaction obeys a ping-ong mechanism. Site-directed mutagenesis was also performed on hTPK1 to examine the contributions of eight strictly conserved residues in thiamin pyrophosphokinase on the kinetic properties. Mutations D71N, D73N, and D100N reduced kcat markedly, indicating that these aspartic acids play a crucial role in carrying out the catalytic process of hTPK1. A selective decrease in the kcat/Kmthiamin value was observed in the D133N mutant, whereas the kcat/KmATV values of T99A and R131G were significantly decreased. Interestingly, the replacement of Gln-96 with Glu caused an increase in the kcat/Kmthiamin value (3.53-fold of the wild-type). It was therefore suggested that the residues Gln-96, Thr-99, Arg-131, and Asp-133 are conserved as functionally significant components for substrate recognition in thiamin pyrophosphokinase.
- 財団法人 学会誌刊行センターの論文
- 2003-06-01
著者
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ONOZUKA Mari
Department of Biochemistry and Molecular Biology, Kyoto Prefectural University of Medicine
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Onozuka Mari
Department Of Biochemistry Kyoto Prefectural University Of Medicine
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Nosaka Kazuto
Department Of Chemistry Graduate School Of Medical Science Kyoto Prefectural University Of Medicine
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Nosaka Kazuto
Department Of Chemistry Kyoto Prefectural University Of Medicine
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