Kinetic studies of the predicted substrate-binding site of varicella-zoster virus thymidine kinase

Abstract

To investigate the mechanism of kinetic action and substrate recognition of varicella-zoster virus (VZV) thymidine kinase (TK), we designed and isolated a sitedirected mutant VZV TK which has double amino acid substitutions, 136threonine to leucine and la7isoleucine to leucine (SDM TK). This mutant was designed to alter the substrate-binding site of the VZV TK to duplicate that of the herpes simplex virus type 2 enzyme. Kinetic studies of the activity of wild-type TK indicated that the binding order of ATP and thymidine is random and that wild-type VZV TK possessed high thymidylate kinase (TM-K) activity. The sensitivity of VZV TK to bisubstrate analogues, dinucleotides of adenosine and thymidine, showed that the optimum distance between the ATP-and substrate-binding sites is two phosphoryl groups greater than with the natural substrate for TK activity. SDM TK lost deoxycytidine kinase activity and had reduced TK and TM-K activities. Inhibition studies on both WT and SDM TK by 5-halogenovinyluridine analogues and their 5' monophosphate derivatives revealed that amino acids at positions 136 and 137 are involved in substrate binding, probably through a role in the formation of the binding pocket for bulky substrates.

Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes

“…ACV and BV-araU are phosphorylated by VZV TK (1,23), but the affinities between these nucleosides and VZV TK are quite different. The K i value of ACV for VZV TK is Ͼ500 M, and that of BV-araU is 0.26 M (17). Moreover, the affinities of these nucleoside triphosphates for cellular DNA polymerase also differ, and the K i values of ACV triphosphate and BV-araU triphosphate are 2.1 M (6) and 0.007 to 0.14 M (14), respectively.…”

Carboranyl thymidine analogues for neutron capture therapy

“…TKs from herpes viruses comprise a third group of dNKs with amino acid sequences and overall structures more closely related to the enzymes of the non-TK1-like family 60,98,[109][110][111][112] Dual TKand TMPK activity is a special feature of most herpes viruses TKs. [112][113][114][115] Recent crystallographic studies with BaTK and BcTK, having 96% amino acid sequence identity, 99 have shown that depending on the presence or absence of the endogenous substrate, Thd, in the substrate binding pocket, TK1-like enzymes exist in a closed and a more open form (Fig. 4).…”

Mutations Distal to the Substrate Site Can Affect Varicella Zoster Virus Thymidine Kinase Activity: Implications for Drug Design

“…The thymidine K m value for wild-type VZVTK used as a reference was 1.96 M (Table 1) compared with literature K m values of between 0.3 and 0.6 M (Suzutani et al, 1993;Amrhein et al, 2000). Whereas our K m value was greater than the average, VZVTK wildtype and mutants were purified, and their activity was as- (Table 1), with the R84V thymidine kinase mutant showing the weakest apparent affinity (9.1 Ϯ 1.5 M).…”

“…The previously reported site-directed mutagenesis of residues making direct contact with the thymidine pyrimidine ring invariably led to almost complete loss of enzyme activity, even when substituting HSV1TK residues into VZVTK (Roberts et al, 1991;Suzutani et al, 1993). Because it was necessary to retain significant enzyme activity, we tried a more subtle approach.…”