Steady-State Kinetics and Mutational Studies of Recombinant Human Thiamin Pyrophosphokinase

Abstract

SummaryThiamin 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/Kmthiamin value was observed in the D133N mutant , whereas the kcat/KmATP 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.

Pyrithiamine as a Substrate for Thiamine Pyrophosphokinase

“…In fact, a recent study suggested that UTP is a better substrate than ATP and the specific activity of TPK at fixed concentration of different nucleotides followed the following sequence UTPϾATPϾCTPϾGTP (29). This is an interesting finding and is consistent with our structure since the interactions surrounding the adenine ring in TPK would be better suited in terms of hydrogen bond donor and acceptor orientations for uracil or GTP.…”

“…Our ternary complex structure is at odds with recent kinetic experiments that suggested TPK obeys a ping-pong mechanism in which Mg 2ϩ /ATP binds first and the product AMP is released with retention of pyrophosphate on the enzyme (29). Subsequent to the release of AMP, thiamine binds, the pyrophosphate is transferred to thiamine, and the product thiamine pyrophosphate is released.…”

“…These experiments probed the catalytic impact of residue exchanges at positions 71, 73, 74, 96, 99, 100, 131, and 133 (29) to Asn all rendered the enzyme virtually inactive, consistent with their role in properly binding and modifying the ligand field of the metal ion for stabilization of the transition state. Mutation of the remaining residues was associated with modest changes in kinetic parameters that can be reconciled based on the ternary complexes presented here.…”

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

“…The K m values of the two AtTPKs fell between previously reported plant TPK K m values of 0.15 lM for parsley (Mitsuda et al 1979) and 4.64 lM for soybean . In contrast to plant TPKs, the human TPK has much lower affinity for thiamin with a K m of 0.21 mM (Onozuka and Nosaka 2003). From these combined analyses, it appears that the AtTPKs are catalytically very similar to each other and to other previously studied plant TPKs.…”

Molecular Cloning and Analysis of the 5'-Flanking Region of the Human Thiamin Pyrophosphokinase Gene

“…Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin to TDP in the presence of ATP and Mg2+, and this enzyme is absolutely required for TDP synthesis. We demonstrated that human thiamin pyrophosphokinase (hTPK1) activity is regulated by the concentration of ATP relative to that of Mg2+ (3). We also isolated an hTPK1 cDNA and as signed the chromosomal localization of the gene to 7834 (4).…”