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标题：Steady-State Kinetics and Mutational Studies of Recombinant Human Thiamin Pyraophosphokinase
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作者：Mari ONOZUKA ; Kazuto NOSAKA
期刊名称：Journal of Nutritional Science and Vitaminology
印刷版ISSN：0301-4800
电子版ISSN：1881-7742
出版年度：2003
卷号：49
期号：3
页码：156-162
DOI：10.3177/jnsv.49.156
出版社：Center for Academic Publications Japan
摘要：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 k_cat markedly, indicating that these aspartic acids play a crucial role in carrying out the catalytic process of hTPK1. A selective decrease in the k_cat /Km^thiamin value was observed in the D133N mutant, whereas the k_cat /Km^ATV values of T99A and R131G were significantly decreased. Interestingly, the replacement of Gln-96 with Glu caused an increase in the k_cat /Km^thiamin 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.
关键词：thiamin pyrophosphokinase;thiamin;thiamin pyrophosphate;steady-state kinetics;site-directed mutagenesis