Abstract:
:The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Pettigrew DW,Smith GB,Thomas KP,Dodds DCdoi
10.1006/abbi.1997.0444subject
Has Abstractpub_date
1998-01-15 00:00:00pages
236-45issue
2eissn
0003-9861issn
1096-0384pii
S0003-9861(97)90444-6journal_volume
349pub_type
杂志文章abstract::Interactions between loops 2, 2' and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall c...
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pub_type: 杂志文章
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pub_type: 杂志文章,评审
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:2006-11-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2003.11.013
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2013.03.013
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(91)90033-f
更新日期:1991-04-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:2004-08-01 00:00:00
abstract::Atroxase, isolated from the venom of Crotalus atrox (western diamondback rattlesnake), is a nonhemorrhagic protease which has fibrinolytic activity in vivo. The primary structure of atroxase was deduced from the cDNA encoding the atroxase protein. The venom glands of Crotalus atrox were used to prepare a cDNA library....
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1995.1175
更新日期:1995-03-10 00:00:00