Abstract：
:Using a combination of intrinsic fluorescence to report ATP-induced rearrangements, quenched-flow to measure ATP hydrolysis "on-enzyme" and optical methods to probe the kinetics of product release, we have begun to dissect the process of energy transduction in the thermosome, a type II chaperonin from Thermoplasma acidophilum. Stoichiometric measurements of ATP binding reveal the tight association of eight nucleotide molecules per hexa-decamer, implying the filling of only one ring owing to strong negative cooperativity. After binding, we show that these eight ATP molecules are hydrolysed over the next 50 s, after which hydrolysis slows down markedly during the establishment of the steady state in the ATPase reaction, demonstrating that the kinetic system is off-rate limited. Looking in more detail, this rapid first-turnover can be dissected into two phases; the first occurring with a half-time of 0.8 s, the second with a half-time of 14 s, possibly reflecting the differential behaviour of the four alpha and four beta subunits in a single thermosome ring. To investigate the post-hydrolytic events, we used two heat-stable enzyme-linked optical assays to measure the rate of evolution of ADP and of phosphate from the thermosome active site. Neither product showed a rapid dissociation phase prior to the establishment of the steady state, showing that both are released slowly at a rate that limits the cycle. These data highlight the importance of the highly populated thermosome/ADP/Pi complex in the molecular mechanism.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Bigotti MG,Bellamy SR,Clarke ARdoi
10.1016/j.jmb.2006.07.064subject
Has Abstractpub_date
2006-09-29 00:00:00pages
835-43issue
4eissn
0022-2836issn
1089-8638pii
S0022-2836(06)00961-2journal_volume
362pub_type
杂志文章abstract：:The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(92)90838-b
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(85)90429-2
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/S0022-2836(02)00246-2
更新日期：2002-05-24 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1996.0880
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abstract：:Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 Å resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3(...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2010.08.023
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1993.1464
更新日期：1993-08-20 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.05.052
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1998.1904
更新日期：1998-07-24 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2006.11.018
更新日期：2007-02-09 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2004-12-10 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(91)90215-r
更新日期：1991-11-20 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(87)90633-4
更新日期：1987-01-05 00:00:00
abstract：:A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermo...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(86)90438-9
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journal_title：Journal of molecular biology
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更新日期：2020-05-01 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2007.03.018
更新日期：2007-06-08 00:00:00
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journal_title：Journal of molecular biology
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2009-01-23 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2012-04-13 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：1990-11-20 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2008-10-17 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2010.06.054
更新日期：2010-09-03 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2002-02-01 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1996.0375
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：1999-12-17 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：1990-10-20 00:00:00
abstract：:Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence t...
journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2015-03-13 00:00:00
abstract：:Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing conti...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2017.06.025
更新日期：2017-09-01 00:00:00
