Thiamine, Biosynthesis and Function

Leder, Irwin G.

doi:10.1016/b978-0-12-299257-5.50010-5

Cited by 8 publications

(4 citation statements)

References 153 publications

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“…On the other hand, the presence of the mutants with markedly reduced activities of both Th-kinase and TMP-PPase described here raises a question. Since TMP-PPase has been established to be an essential enzyme for thiamine biosynthesis in S. cerevisiae (9), a marked decrease in this enzyme activity might affect the yeast growth in minimal medium. However, this is not the case in the mutants isolated in this study, but the small enzyme activity remaining appears to afford the cellular requirement for thiamine.…”

Section: Discussionmentioning

confidence: 99%

Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway

Kawasaki

1993

J Bacteriol

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Mutants of Saccharomyces cerevisiae resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole, an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), which are deficient in the activities of both hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase, involved in the pathway of de novo synthesis of thiamine in S. cerevisiae, have been isolated. Genetic analysis revealed that the mutation occurs at a single gene in the nucleus. The two enzyme activities were copurified to apparent homogeneity, and the molecular masses of the purified proteins were found to be approximately 470 and 60 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Hydroxyethylthiazole kinase was specific for ATP and Mg2+, although to a lesser extent a combination with other nucleoside triphosphates or divalent cations could replace them. p-Chloromercuribenzoate was a potent inhibitor of the enzyme, and the inhibition was prevented by the addition of 2-mercaptoethanol. These findings indicate that yeast hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity, which is an octamer of identical 60-kDa subunits.

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Section: Discussionmentioning

confidence: 99%

Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway

Kawasaki

1993

J Bacteriol

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“…TDP is a cofactor for many enzymes indispensable for glucose and energy metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, transke tolase, and others (1,2). The primary structures and conformations of many of these TDP-dependent enzymes have been well studied, and the mechanism and configuration of TDP as a transient intermediate carrier of the aldehyde group in the enzymes have been characterized (3).…”

mentioning

confidence: 99%

“…Thiamin occurs in cells as free thiamin and as its phosphate esters, thiamin monophosphate, thiamin pyrophosphate (TDP), and thiamin triphosphate (1,2). TDP is a cofactor for many enzymes indispensable for glucose and energy metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, transke tolase, and others (1,2).…”

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confidence: 99%

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

Onozuka

Nosaka

2003

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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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.

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“…The existence of thiamine triphosphate in brain and other tissues has stimulated numerous studies, and the possibility of a direct role of thiamine in neural excitation has been considered (Leder, 1975).…”

Section: Imentioning

confidence: 99%