Steady-state kinetics and mutational studies of recombinant human thiamin pyrophosphokinase.
Thiamin pyrophosphokinase catalyzes the pyrophosphorylation of thiamin to thiamin pyrophosphate in the presence of ATP and Mg2+. 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-pong 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/Km(thiamin) value was observed in the D133N mutant, whereas the kcat/Km(ATP) values of T99A and R131G were significantly decreased. Interestingly, the replacement of Gln-96 with Glu caused an increase in the kcat/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.
Onozuka
Mari
M
Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan.
Nosaka
Kazuto
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D016415:Sequence Alignment; D013836:Thiamin Pyrophosphokinase / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics*; D013831:Thiamine / Q000378:metabolism
D011994:Recombinant Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D013836:Thiamin Pyrophosphokinase; D013831:Thiamine