Kinetics mechanism and regulation of native human hepatic thymidine phosphorylase

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Abstract

Thymidine phosphorylase (TP; EC 2.4.2.4) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective nucleobases and 2-deoxy-α-d-ribose-1-phosphate (dRib-1-P). TP is a key enzyme in the pyrimidine salvage pathways. Activity of the enzyme is crucial in angiogenesis, cancer chemotherapy, radiotherapy, and tumor imaging, Nevertheless, a complete set of kinetic parameters has never been reported for any human TP. This study describes the kinetic mechanism and regulation of native human hepatic TP. The liver is a main site of pyrimidine metabolism and contains high levels of TP. Initial velocity and product inhibition studies demonstrated that the basic mechanism of this enzyme is a sequential random bi-bi mechanism. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation, and a binding pattern indicating that the binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KThymidine = 284 ± 55, KPi = 5.8 ± 1.9, KThymine = 244 ± 69, and KdRib-1-P = 90 ± 33 μM. Thymine was a product activator, but becomes a substrate inhibitor at concentrations eight times higher than its Km. dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bounded better to the Enzyme●Pi complex than the free enzyme, but had better affinity to the free enzyme than the Enzyme●Thymidine complex. In the reverse reaction, dRib-1-P enhanced the binding of thymine. The enhancement of the thymine binding along with the fact that dRib-1-P was a non-competitive product inhibitor suggests the presence of another binding site for dRib-1-P on the enzyme.

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.

References (41)

  • E. Boschetti et al.

    Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy

    PLoS One

    (2014)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal. Biochem.

    (1976)
  • N.S. Brown et al.

    Thymidine phosphorylase, 2-deoxy-d-ribose and angiogenesis

    Biochem. J.

    (1998)
  • W.W. Cleland

    The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations

    Biochim. Biophys. Acta

    (1963)
  • W.W. Cleland

    The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory

    Biochim. Biophys. Acta

    (1963)
  • W.W. Cleland

    The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection

    Biochim. Biophys. Acta

    (1963)
  • W.W. Cleland

    The statistical analysis of enzyme kinetic data

    Adv. Enzymol.

    (1967)
  • C. Deves et al.

    The kinetic mechanism of human thymidine phosphorylase -A molecular target for cancer drug development

    Mol. Biosyst.

    (2014)
  • M.H. el Kouni et al.

    Uridine phosphorylase from Schistosoma mansoni

    J. Biol. Chem.

    (1988)
  • M.H. el Kouni et al.

    Differences in activities and substrate specificities of human and murine pyrimidine nucleoside phosphorylases: implications for chemotherapy with 5-fluoropyrimidines

    Cancer Res.

    (1993)
  • W.D. Ensminger et al.

    A clinical-pharmacological evaluation of hepatic arterial infusions of 5-fluoro-2’-deoxyuridine and 5-fluorouracil

    Cancer Res.

    (1978)
  • T.-P. Fan et al.
  • J. Folkman

    What is the evidence that tumors are angiogenesis dependent?

    J. Natl. Cancer Inst.

    (1990)
  • S.B. Fox et al.

    The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase is up-regulated in breast cancer epithelium and endothelium

    Br. J. Cancer

    (1996)
  • T. Furukawa et al.

    Angiogenic factor

    Nature

    (1992)
  • R.C. Gallo et al.

    The enzymatic mechanisms for deoxythymidine synthesis in human leukocytes

    J. Biol. Chem.

    (1967)
  • M. Haraguchi et al.

    Angiogenic activity of enzymes

    Nature

    (1994)
  • B. Higley et al.

    Pyrimidine nucleoside phosphorylase activity in tumor and matched normal gastrointestinal mucosa

    Gut

    (1982)
  • T. Hotta et al.

    Increased expression of thymidine phosphorylase in tumor tissue in proportion to TP-expression in primary normal tissue

    Oncol. Rep.

    (2004)
  • M.H. Iltzsch et al.

    Kinetic studies of thymidine phosphorylase from mouse liver

    Biochemistry

    (1985)
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      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).

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