Interaction of the Recombinant Herpes Simplex Virus Type 1 Thymidine Kinase with Thymidine and Aciclovir: A Kinetic Study

Kussmann-Gerber, Susanna; Wurth, Christine; Scapozza, Léonardo; Pilger, Beatrice; Pliška, V.; Folkers, Gerd

doi:10.1080/15257779908043078

Cited by 12 publications

(12 citation statements)

References 22 publications

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“…Moreover, the radiometric results show a K m value of n-MCT corresponding to the K i value of 11.4 Ϯ 1.6 M published by Prota et al (17). This confirms that the K i is equal to the K m in the case of HSV-1 TK, as reported earlier (17,19,20).…”

Section: N-mct Radiometric Assaysupporting

confidence: 91%

“…New (7); c radiometric study (19); d radiometric study (21); e radiometric study (20); f binding affinities reported as K i in Griffiths (27). In the case of HSV-1 TK, K m and K i values can both be used as binding affinity constant because K m is equal to K i (20).…”

Section: Easy Determination Of the K Cat Values Of Antiviral Lead Commentioning

confidence: 97%

“…In the case of HSV-1 TK, K m and K i values can both be used as binding affinity constant because K m is equal to K i (20). n.d., not determined.…”

Section: Easy Determination Of the K Cat Values Of Antiviral Lead Commentioning

confidence: 99%

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A Spectrophotometric Assay for Quantitative Determination of kcat of Herpes Simplex Virus Type 1 Thymidine Kinase Substrates

Schelling¹,

Folkers²,

Scapozza³

2001

Analytical Biochemistry

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Section: N-mct Radiometric Assaysupporting

confidence: 91%

Section: Easy Determination Of the K Cat Values Of Antiviral Lead Commentioning

confidence: 97%

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A Spectrophotometric Assay for Quantitative Determination of kcat of Herpes Simplex Virus Type 1 Thymidine Kinase Substrates

Schelling¹,

Folkers²,

Scapozza³

2001

Analytical Biochemistry

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“…Table I shows that the measured k cat of dT agrees with those reported in literature (31,32). Also the k cat of ACV corresponds to that measured in two independent laboratories (33,34). In the literature a value of 0.015 s Ϫ1 is also reported for ACV (31), but, considering the newly reported values, this seems to be an outliner.…”

Section: Catalytic Activity Of Tk Hsv1supporting

confidence: 76%

The Rational of Catalytic Activity of Herpes Simplex Virus Thymidine Kinase

Sulpizi

Schelling²,

Folkers³

et al. 2001

Journal of Biological Chemistry

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Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK HSV1 ) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK HSV1 ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK HSV1 -ligand complex is available. The k cat values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu 225 . A striking correlation is found between the energetics associated with this interaction and the k cat values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK HSV1 -linked suicide gene therapies.

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“…In our attempt to rationalize this property, we have designed a series of (47)(48)(49). This represents a peculiarity of HSV 1 TK by which dT is binding prior to ATP, and the rate constant of disintegration of the intermediate complex (ES) is negligible as compared to the corresponding dissociation rate constant (46).…”

Section: Discussionmentioning

confidence: 99%

Substrate Diversity of Herpes Simplex Virus Thymidine Kinase

Pilger¹,

Perozzo²,

Alber³

et al. 1999

Journal of Biological Chemistry

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Herpes simplex virus type 1 (HSV 1) 1 thymidine kinase (TK) is a multifunctional enzyme that possesses kinase activities normally performed by three separate cellular enzymes. It phosphorylates thymidine (dT), which is then transformed by cellular kinases to the triphosphorylated DNA building block, and deoxyuridine (dU); both reactions are comparable to the function of human cellular TK. Further, it converts deoxycytidine (dC) to dCMP, as does human deoxycytidine kinase (dCK), and phosphorylates thymidylate (dTMP), as does human TMP kinase (TmpK) (1-3). Moreover, unlike its cellular counterpart human cellular TK, HSV 1 TK is able to phosphorylate pyrimidine, as well as purine analogs, and discloses low stereochemical demands for the ribose moiety, as it also accepts acyclic side chains as phosphoryl group acceptors e.g. (4 -6). These differences in substrate diversity are the crucial molecular basis for the selective treatment of viral infections. Nowadays, the most widely used therapeutic compounds to interfere with a severe HSV 1 infection are the purine analogs acyclovir (ACV) and penciclovir and their prodrugs valaciclovir and famciclovir, respectively. They require HSV 1 TK to be efficiently activated in order to block virus proliferation by inhibition of viral DNA polymerase. HSV 1 TK is the key enzyme in this antiviral strategy. In gene therapy of cancer (7,8) and AIDS (9), HSV 1 TK is used as a suicide enzyme in combination with the purine analog ganciclovir. Another important application is the use of HSV 1 TK as a rescue system in allogeneic bone marrow transplantation-induced graft versus host disease (10). In addition to the significance from a therapeutic point of view, HSV 1 TK seems to be important for the reactivation of the virus from lifelong latent infection in neuronal ganglia (11-13). However, there is evidence that human TK can functionally replace viral TK in terms of reactivation of the virus from latency (14).There are no recognizable sequence similarities between HSV 1 TK and human cellular TK (15). Rather, sequence alignments have detected similarities between herpesvirus TKs and human dCK (16) and to a lesser extent cellular TmpK (17). Despite the limited sequence homology with enzymes of the nucleotide kinase (NK) family, HSV 1 TK shares structural features comprising a parallel five-stranded ␤-sheet and a glycine-rich loop common to all NKs. In the crystal structure, HSV 1 TK is a homodimeric enzyme with 376 amino acids per subunit (18 -20). The two subunits are related by C2 symmetry. The active site is formed by an ATP-and a nucleoside-binding region. The visual representation of the thymidine binding site is depicted in Fig. 1, featuring a complex hydrogen bond network within the active site. The thymine ring makes pairwise hydrogen bond interaction via its 4-carbonyl and 3-NH group with the amide group of the highly conserved Gln-125 and hydrogen bonds with Arg-176 by means of two ordered water molecules. Moreover, the pyrimidine ring of thymidine is fixed between Met-128 and T...

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Interaction of the Recombinant Herpes Simplex Virus Type 1 Thymidine Kinase with Thymidine and Aciclovir: A Kinetic Study

Cited by 12 publications

References 22 publications

A Spectrophotometric Assay for Quantitative Determination of kcat of Herpes Simplex Virus Type 1 Thymidine Kinase Substrates

A Spectrophotometric Assay for Quantitative Determination of kcat of Herpes Simplex Virus Type 1 Thymidine Kinase Substrates

The Rational of Catalytic Activity of Herpes Simplex Virus Thymidine Kinase

Substrate Diversity of Herpes Simplex Virus Thymidine Kinase

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