Polyphosphatase activity of CthTTM, a bacterial triphosphate tunnel metalloenzyme Journal Article
| Authors: | Jain, R.; Shuman, S. |
| Article Title: | Polyphosphatase activity of CthTTM, a bacterial triphosphate tunnel metalloenzyme |
| Abstract: | Triphosphate tunnel metalloenzymes (TTMs) are a superfamily of phosphotransferases with a distinctive active site located within an eight-stranded β barrel. The best understood family members are the eukaryal RNA triphosphatases, which catalyze the initial step in mRNA capping. The RNA triphosphatases characteristically hydrolyze nucleoside 5′-triphosphates in the presence of manganese and are inept at cleaving inorganic tripolyphosphate. We recently identified a TTM protein from the bacterium Clostridium thermocellum (CthTTM) with the opposite substrate preference. Here we report that CthTTMcatalyzes hydrolysis of guanosine 5′-tetraphosphate to yield GTP and Pi (Km = 70 μM, k cat = 170 s-1) much more effectively than it converts GTP to GDP and Pi (Km = 70 μM, kcat = 0.3 s -1), implying that a nucleoside interferes when positioned too close to the tunnel entrance. CthTTM is capable of quantitatively cleaving diadenosine hexaphosphate but has feeble activity with shorter derivatives diadenosine tetraphosphate and diadenosine pentaphosphate. We propose that the tunnel opens to accommodate the dumbbell-shaped diadenosine hexaphosphate and then closes around it to perform catalysis. We find that CthTTM can exhaustively hydrolyze a long-chain inorganic polyphosphate, a molecule that plays important roles in bacterial physiology. CthTTM differs from other known polyphosphatases in that it yields a ∼2:1 mixture of Pi and PPi end products. Bacterial/archaeal TTMs have a C-terminal helix located near the tunnel entrance. Deletion of this helix from CthTTM exerts pleiotropic effects. (i) It suppresses hydrolysis of guanosine 5′-tetraphosphate and inorganic PPPi; (ii) it stimulates NTP hydrolysis; and (iii) it biases the outcome of the longchain polyphosphatase reaction more strongly in favor of Pi production. We discuss models for substrate binding in the triphosphate tunnel. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; unclassified drug; nonhuman; outcome assessment; protein analysis; phosphatase; carboxy terminal sequence; bacteria (microorganisms); rna; bacterial proteins; guanosine; eukaryota; quantitative analysis; nucleic acids; substrate specificity; models, molecular; binding sites; catalysis; guanosine triphosphate; hydrolysis; protein structure, secondary; phosphotransferases; pleiotropy; guanosine diphosphate; bacteriology; family members; bacterial enzyme; clostridium thermocellum; manganese; pleiotropic effects; vehicular tunnels; active sites; end products; metalloenzyme; polyphosphate; substrate bindings; terminal helixes; triphosphate; triphosphate tunnel metalloenzymes; tripolyphosphate; tunnels; diadenosine hexaphosphate; diadenosine pentaphosphate; diadenosine tetraphosphate; guanosine 3' diphosphate 5' diphosphate; polyphosphatase; triphosphate tunnel metalloenzyme; metalloproteins; nucleoside-triphosphatase |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 283 |
| Issue: | 45 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2008-11-07 |
| Start Page: | 31047 |
| End Page: | 31057 |
| Language: | English |
| DOI: | 10.1074/jbc.M805392200 |
| PUBMED: | 18782773 |
| PROVIDER: | scopus |
| PMCID: | PMC2576530 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 17 November 2011" - "CODEN: JBCHA" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work