Kinetics mechanism and regulation of native human hepatic thymidine phosphorylase
Introduction
Thymidine phosphorylase (TP; EC 2.4.2.4) is an important enzyme of the pyrimidine salvage pathways. It catalyzes the reversible phosphorolysis of the pyrimidine deoxyribosides; thymidine, deoxyuridine, but not deoxycytidine, and their analogues to their respective nucleobases and 2-deoxy-α-d-ribose-1-phosphate (dRib-1-P) as follows:Pyrimidine deoxyriboside + Pi ⇋ Pyrimidine nucleobase + dRib-1-P
TP activity is also an essential step in the regulation of intra- or extracellular thymidine concentration, thymidine homeostasis, and angiogenesis in mammalian cells (Janion and Shugar, 1961; Gallo et al., 1967; Schwartz and Milstone, 1988; Schwartz et al., 1988a; and b; Shaw, 1988; Shaw et al., 1988; Folkman, 1990; Fan et al., 1992; Lees and Fan, 1994; Reynolds et al., 1994; Haraguchi et al., 1994; Moghaddam et al., 1995; Miyadera et al., 1995; Brown and Bicknell, 1998; Uchimiya et al., 2002). The enzyme is identical to the platelet derived-endothelial cell growth factor (PD-ECGF) (Usuki et al., 1994; Furukawa et al., 1992). Mutations in the TP gene are associated with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive human disease exhibiting multiple deletions of skeletal muscle. MNGIE patients accumulate thymidine systemically, which ultimately results in imbalances in the mitochondrial pool of deoxyribonucleoside triphosphates that interferes with mitochondrial DNA replication, and in turn causes mitochondrial dysfunction (Nishino et al., 2000). In addition, TP plays a critical role in cancer chemotherapy, radiotherapy as well as tumor imaging. The expression of the enzyme seems to affect sensitivity of the cell to the pyrimidine analogues, as it activates or deactivates some of most frequently used chemotherapeutic pyrimidine nucleoside analogues (Ensminger et al., 1978; el Kouni et al., 1993; Schuller et al., 2000; Tsukamoto et al., 2000). Furthermore, overexpression of TP has been reported in many primary and metastatic tumors, relative to the surrounding normal tissue (Fox et al., 1996; Higley et al., 1982; Hotta et al. (2004); Imazano et al., 1997). Therefore, specific inhibitors of TP may be useful as chemotherapeutic agents by enhancing the antineoplastic efficacy of some pyrimidine analogues or prevention of angiogenesis and hence tumor growth and metastasis. The search for TP inhibitors could benefit greatly from kinetic studies of the enzyme. Detailed kinetic studies could reveal a great deal about the structure, and function of TP. Such studies are also essential to fully understand the basic reaction mechanism of the enzyme (e.g. ping-pong, sequential, random, ordered, etc.), and to illustrate the order of binding of the substrates and release of the products. The order of addition of substrates and the mechanism of action of the enzyme would shed some light on the topology of the active center and whether there is a “cooperative effect" between the substrates or not, etc. Such information cannot be visualized by x-ray crystallography of the enzyme, but could be critical for interpreting crystallographic results. Thus, kinetic analysis should be a top priority for structure-based strategy for the design, synthesis and evaluation of novel inhibitors of human TP. Nevertheless, a complete set of kinetic parameters has never been achieved for any human TP.
The present study was performed to determine the kinetic parameters of native human hepatic TP. The liver is a major site of pyrimidine metabolism and contains high levels of TP (Ensminger et al., 1978; Kono et al., 1984; Iltzsch et al., 1985; LaCreta et al., 1989; el Kouni et al., 1993; Boschetti et al., 2014). Furthermore, human hepatic TP is also distinct from the enzymes in extrahepatic tissues (e.g. placenta) as well as from the liver of other animals in substrate specificity and other characteristics (el Kouni et al., 1993; Oh and el Kouni, 2018).
Section snippets
Chemicals
[2-14C]thymidine (56 Ci/mol) and [2-14C]thymine (56 Ci/mol) were from Moravek Biochemicals Inc., Brea, CA; Macherey Nagel Polygram Silica Gel G/UV254 thin layer chromatography plates from Fisher cientific, NJ; Bio-Rad protein assay kit, from Bio-Rad Laboratories, Hercules, CA. All other chemicals were obtained from Sigma Chemical Co., St. Louis, MO.
Source of human hepatic TP
Homogenously purified native human hepatic TP was prepared as previously described (Oh and el Kouni, 2018). Protein concentrations were determined
Optimum pH
Maximal enzymatic activities were estimated in a broad range of pH (4.0–11.0) for both the forward (thymidine phosphorolysis), and the reverse (thymidine synthesis) reactions. For thymidine phosphorolysis, maximal activities ranged broadly from pH 5.0 to 8.5 (Fig. 1A). For thymidine synthesis, maximal enzymatic activity occurred at pH 7.5 (Fig. 1B). Therefore, pH 7.5 was chosen as an optimum pH for the kinetic studies.
Thymidine and phosphate as substrates
Initial velocity studies were carried out with varied thymidine (30–720 μM)
Conclusions
This is first complete set of kinetic parameters reported for any human TP. In view of the absence of any uncompetitive pattern of the double reciprocal plots, the enzymatic mechanism is consistent with a rapid equilibrium random sequential bi-bi mechanism. dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bound better to Enzyme●Pi complex than to free enzyme, but has better affinity to free enzyme than to Enzyme●Thymidine complex. On the other hand, dRib-1-P enhanced
Acknowledgments
We Thank Dr. Fardos N. M. Naguib for her help with the computer programing.
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2022, European Journal of PharmacologyCitation Excerpt :However, the mechanism by which TP induces angiogenesis has been not completely understood. Several reports suggested that TP catalyzes the reversible phosphorolysis of thymidine (dThd) and its analogs to thymine (1) and 2-deoxy-α-D-ribose-1-phosphate (2) (Fig. 1) (Nencka et al., 2005; Oh and el Kouni, 2019; Reigan et al., 2004). The latter product dephosphorylates to produce 2-deoxy-α-D-ribose, which displayed endothelial cell chemotactic activity and can promote angiogenesis (Ackland and Peters, 1999; Miwa et al., 1998).