Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation Journal Article
| Authors: | Samai, P.; Shuman, S. |
| Article Title: | Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation |
| Abstract: | Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3′-OH and 5′-PO4 termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PPi and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5′-phosphate to form DNA-adenylate; 3) the 3′-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg2+. Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (kstep3 = 25 s-1) exceeds that for DNA adenylylation (kstep2 = 2.4 s-1) and that Mg2+ binds with similar affinity during step 2 (Kd = 0.77 mM) and step 3 (Kd = 0.87 mM). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5′-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5′-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; protein expression; missense mutation; binding affinity; chemical analysis; carboxy terminal sequence; protein dna binding; ph; enzyme activity; dna; amino acid sequence; kinetics; messenger rna; 5' untranslated region; crystal structure; 3' untranslated region; mutation rate; catalysis; kinetic analysis; structure analysis; polydeoxyribonucleotide synthase; dna determination; enzymes; rate constants; proton transport; enzyme mechanism; esters; dna ligases; enzyme active site; mrna capping; magnesium ion; phosphorus; divalent cation; functional groups; adenylation; nucleotidyltransferase; c terminus; dna strand; mechanistic studies; dna strands; viruses; adenylylation; phosphodiesters; polynucleotides; virus enzyme; ph measurement; protonation; carboxylation; burst kinetics; instructive models; pre-steady state; rate limiting |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 287 |
| Issue: | 34 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2012-08-17 |
| Start Page: | 28609 |
| End Page: | 28618 |
| Language: | English |
| DOI: | 10.1074/jbc.M112.380428 |
| PROVIDER: | scopus |
| PMCID: | PMC3436572 |
| PUBMED: | 22745124 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 1 October 2012" - "CODEN: JBCHA" - "Source: Scopus" |
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