Insights into product release dynamics through structural analyses of thymidylate kinase

Chaudhary, Santosh Kumar; Iyyappan, Yuvaraj; Elayappan, Mohanapriya; Jeyakanthan, Jeyaraman; Sekar, K.

doi:10.1016/j.ijbiomac.2018.11.025

Cited by 6 publications

(2 citation statements)

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“…The ADP titration suggests that F105Y hTMPK has increased affinity for ADP, and Ile184δ1 shows no exchange for both 2 and 8 mM samples of either F105Y complex. Human TMPK has been shown to follow a random bi–bi mechanism suggesting that the order of product release is random; however, a recent random accelerated molecular dynamics study of WT hTMPK suggests that TDP release pathways are largely biased toward the residues immediately following α6 in the LID whether or not ADP had already dissociated . This suggests slower ADP release may be responsible for some loss in activity of the F105Y enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…Human TMPK has been shown to follow a random bi−bi mechanism suggesting that the order of product release is random; however, a recent random accelerated molecular dynamics study of WT hTMPK suggests that TDP release pathways are largely biased toward the residues immediately following α6 in the LID whether or not ADP had already dissociated. 41 This suggests slower ADP release may be responsible for some loss in activity of the F105Y enzyme. This region is stabilized in the F105Y complexes, as reported on by Leu137δ1.…”

Section: ■ Discussionmentioning

confidence: 99%

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Stabilization of Active Site Dynamics Leads to Increased Activity with 3′-Azido-3′-deoxythymidine Monophosphate for F105Y Mutant Human Thymidylate Kinase

2020

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Thymidylate kinases are essential enzymes with roles in DNA synthesis and repair and have been the target of drug development for antimalarials, antifungals, HIV treatment, and cancer therapeutics. Human thymidylate kinase (hTMPK) conversion of the anti-HIV prodrug 3′-azido-3′-deoxythymidine (AZT or zidovudine) monophosphate to diphosphate is the ratelimiting step in the activation of AZT. A point mutant (F105Y) has been previously reported with significantly increased activity for the monophosphate form of the drug [3′-azidothymidine-5′monophosphate (AZTMP)]. Using solution nuclear magnetic resonance (NMR) techniques, we show that while the wild-type (WT) and F105Y hTMPK adopt the same structure in solution, significant changes in dynamics may explain their different activities toward TMP and AZTMP. 13 C spin-relaxation measurements show that there is little change in dynamics on the ps to ns time scale. In contrast, methyl 1 H relaxation dispersion shows that AZTMP alters adenosine nucleotide handling in the WT protein but not in the mutant. Additionally, the F105Y mutant has reduced conformational flexibility, leading to an increase in affinity for the product ADP and a slower rate of phosphorylation of TMP. The dynamics at the catalytic center for F105Y bound to AZTMP are tuned to the same frequency as WT bound to TMP, which may explain the mutant's catalytic efficiency toward the prodrug.

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Section: Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%