Computational studies of the domain movement and the catalytic mechanism of thymidine phosphorylase

Rick, Steven W.; Abashkin, Yuri G.; Hilderbrandt, R. L.; Burt, Stanley K.

doi:10.1002/(sici)1097-0134(19991101)37:2<242::aid-prot9>3.0.co;2-5

Cited by 27 publications

(22 citation statements)

References 48 publications

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“…Domain movement (once the active site is occupied) is considered to be essential in bringing the substrates into close proximity for the reaction to proceed and has been proposed for other members of the PyNP superfamily as well. Such a movement depending on the bound and unbound state of the enzyme, has also been supported based on computational work on EcTP [21]. Structural alignment of bound (1TPT) and unbound (2TPT) EcTP yielded an rmsd of 0.52 Å for 440 Cα atoms.…”

Section: Resultsmentioning

confidence: 88%

Structures of native human thymidine phosphorylase and in complex with 5-iodouracil

Mitsiki¹,

Papageorgiou²,

Iyer³

et al. 2009

Biochemical and Biophysical Research Communications

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Thymidine phosphorylase (TP) first identified as platelet derived endothelial cell growth factor (PD-ECGF) plays a key role in nucleoside metabolism. Human TP (hTP) is implicated in angiogenesis and is overexpressed in several solid tumors. Here, we report the crystal structures of recombinant hTP and its complex with a substrate 5-iodouracil (5IUR) at 3.0 and 2.5 Å, respectively. In addition, we provide information on the role of specific residues in the enzymatic activity of hTP through mutagenesis and kinetic studies.

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Section: Resultsmentioning

confidence: 88%

Structures of native human thymidine phosphorylase and in complex with 5-iodouracil

Mitsiki¹,

Papageorgiou²,

Iyer³

et al. 2009

Biochemical and Biophysical Research Communications

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“…It is assumed that a domain closure of the cleft is necessary to generate the active site of the enzyme. This domain closure has been simulated by molecular modeling studies and the catalytic mechanism of the enzyme has been proposed (36,37). Very recently, the structure of the human TPase has been revealed in complex with a pyrimidine derivative, TPI that appears to mimic the substrate transition state (15).…”

Section: Discussionmentioning

confidence: 99%

The Nucleoside Derivative 5′-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action

Liekens

Hernández

Ribatti

et al. 2004

Journal of Biological Chemistry

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Thymidine phosphorylase (TPase) catalyzes the reversible phosphorolysis of pyrimidine deoxynucleosides to 2-deoxy-D-ribose-1-phosphate and their respective pyrimidine bases. The enzymatic activity of TPase was found to be essential for its angiogenesis-stimulating properties. All of the previously described TPase inhibitors are either pyrimidine analogues that interact with the nucleosidebinding site of the enzyme or modified purine derivatives that mimic the pyrimidine structure and either compete with thymidine or act as a multisubstrate (competitive) inhibitor. We now describe the inhibitory activity of the purine riboside derivative KIN59 (5 -O-tritylinosine) against human and bacterial recombinant TPase and TPase-induced angiogenesis. In contrast to previously described TPase inhibitors, KIN59 does not compete with the pyrimidine nucleoside or the phosphate-binding site of the enzyme but noncompetitively inhibits TPase when thymidine or phosphate is used as the variable substrate. In addition, KIN59 was far more active than other TPase inhibitors, previously tested by us, against TPase-induced angiogenesis in the chorioallantoic membrane assay. The observed anti-angiogenic effect of KIN59 was not accompanied by inflammation or any visible toxicity. Inosine did not inhibit the enzymatic or angiogenic activity of the enzyme, indicating that the 5 -O-trityl group in KIN59 is essential for the observed effects. In contrast with current concepts, our data indicate that the angiogenic activity of TPase is not solely directed through its functional nucleoside and phosphate-binding sites. Other regulatory (allosteric) site(s) in TPase may play an important role in the mechanism of TPase-triggered angiogenesis stimulation and apoptosis inhibition. Identification of these site(s) is important to obtain a better insight into the molecular role of TPase in the progression of cancer and angiogenic diseases.Angiogenesis is the process by which new blood vessels arise from pre-existing vessels (1). It is essential during embryogenesis for the formation of a vascular plexus, which ensures an adequate blood supply to all developing tissues. In adults, neovascularization is limited to wound healing and the female reproductive cycle. During these processes, angiogenesis is tightly regulated. Unregulated angiogenesis may lead to the progression of several inflammatory diseases and is an essential component of solid tumor growth and metastasis. The formation of new blood vessels requires several sequential steps, which are regulated by a number of angiogenic factors, including chemokines, growth factors, integrins, and enzymes, such as proteases and thymidine phosphorylase (TPase) 1 (1).TPase is an enzyme that catalyzes the reversible phosphorolysis of pyrimidine 2Ј-deoxynucleosides to 2-deoxyribose 1-phosphate and their respective pyrimidine bases. TPase also recognizes several nucleoside analogues that are being used clinically as antiviral

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“…[14][15][16][17][18] In this context, computational chemistry can be an excellent tool in the understanding and interpretation of these kinds of processes and the fundamental chemistry behind them. [19][20][21][22][23][24] There seems to be general agreement on the fact that protonation of the nucleobase catalyzes the hydrolytic cleavage of the N-glycosidic bond by making it a better leaving group, thus facilitating the nucleophilic substitution at the anomeric carbon of the deoxyribose. [25][26][27] The reaction mechanism of this process is still unclear, and much experimental work, especially related to transition state analysis using kinetic isotope effects studies, is being done to clarify it.…”

Section: Introductionmentioning

confidence: 99%

Influence of N7 Protonation on the Mechanism of the N-Glycosidic Bond Hydrolysis in 2‘-Deoxyguanosine. A Theoretical Study

et al. 2007

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The influence of N7 protonation on the mechanism of the N-glycosidic bond hydrolysis in 2'-deoxyguanosine has been studied using density functional theory (DFT) methods. For the neutral system, two different pathways (with retention and inversion of configuration at the C1' anomeric carbon) have been found, both of them consisting of two steps and involving the formation of a dihydrofurane-like intermediate. The Gibbs free energy barrier for the first step is very high in both cases (53 and 46 kcal/mol for the process with inversion and with retention, respectively). However, the N7-protonated system shows a very different mechanism which consists of two steps. The first one leads to the formation of an oxacarbenium ion intermediate, with a Gibbs free energy barrier of 27 kcal/mol, and the second one corresponds to the nucleophilic attack of the water molecule to the oxacarbenium ion and takes place with a barrier of 1.3 kcal/mol. Thus, these results agree with a stepwise SN1 mechanism (DN*AN), with a discrete intermediate formed between the leaving group and the nucleophile approach, and show that N7 protonation strongly catalyzes the hydrolysis of the N-glycosidic bond, making the guanine a better leaving group. Finally, kinetic isotope effects have been calculated for the protonated system, and the results obtained are in very good agreement with experimental data for analogous systems.

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Computational studies of the domain movement and the catalytic mechanism of thymidine phosphorylase

Cited by 27 publications

References 48 publications

Structures of native human thymidine phosphorylase and in complex with 5-iodouracil

Structures of native human thymidine phosphorylase and in complex with 5-iodouracil

The Nucleoside Derivative 5′-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action

Influence of N7 Protonation on the Mechanism of the N-Glycosidic Bond Hydrolysis in 2‘-Deoxyguanosine. A Theoretical Study

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