Consideration of a new catalytic role for the thiazolium sulfur atom of the coenzyme thiamine pyrophosphate

A review of the mechanism of action of thiamine pyrophosphate is presented in brief detail along with a new proposal for the role of the sulfur atom of the thiazolium ring of the coenzyme. Evidence is presented to support the idea that sulfur plays a catalytic role in stabilizing negative charge developing in the transition states of the biological reactions.     

The binding of thiamine pyrophosphate with transketolase in equilibrium conditions

The binding between thiamine pyrophosphate and transketolase, purified from baker's yeast, in equilibrium conditions has been studied. In the presence of Ca²⁺, the enzyme molecule has been shown to possess two binding sites for the coenzyme, whose dissociation constants are 3.2 × 10^?8 and 2.5 × 10^?7M; besides, there are site(s) where the binding of the coenzyme is less firm. In the presence of Mg²⁺, a positive cooperative interaction between the binding sites of thiamine pyrophosphate has been observed. Regardless of the cation used, the major part of the catalytic activity of the transketolase molecule manifests itself in the binding of one molecule of the coenzyme.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg²⁺ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

Cyclopropylglyoxylate as a mechanistic probe of thiamine pyrophosphate dependent pyruvate-metabolizing enzymes

Four pyruvate-decarboxylating enzymes with thiamine pyrophosphate (TPP) cofactors catalyze the decarboxylation of the cyclopropyl substrate analog cyclopropylglyoxylate. Pyruvate: ferredoxin oxidoreductase, an archaebacterial enzyme which catalyzes oxidation of the hydroxyethyl-TPP (HETPP) intermediate by two one-electron transfers to an iron-sulfur center, generates the coenzyme A thioester of cyclopropylcarboxylic acid. A long-lived free radical, HETPP is thought to be an intermediate in the pyruvate to acetyl-CoA conversion; however, cleavage of the cyclopropyl ring was not detected. Pyruvate decarboxylase, pyruvate oxidase, and pyruvate dehydrogenase also generate the corresponding cyclopropyl products. The applicability of cyclopropyl substrate analogs as indicators of free-radical enzyme mechanisms is discussed in light of these results.     

The effect of thiamine and its metabolites on pepsin and trypsin activity

The presence of thiochrome in the incubation medium increases the pepsin activity. Thiamine pyrophosphate decrease the pepsin and trypsin activity. The established facts may be important for the physiological regulation of proteases activity in the stomach-intestine tract.