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| | {{STRUCTURE_1ql6| PDB=1ql6 | SCENE= }} | | {{STRUCTURE_1ql6| PDB=1ql6 | SCENE= }} |
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| - | '''THE CATALYTIC MECHANISM OF PHOSPHORYLASE KINASE PROBED BY MUTATIONAL STUDIES'''
| + | ===THE CATALYTIC MECHANISM OF PHOSPHORYLASE KINASE PROBED BY MUTATIONAL STUDIES=== |
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| - | ==Overview==
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| - | The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis. | + | The line below this paragraph, {{ABSTRACT_PUBMED_10545198}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 10545198 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_10545198}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Serine/threonine-protein]] | | [[Category: Serine/threonine-protein]] |
| | [[Category: Transferase]] | | [[Category: Transferase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 06:24:22 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 13:56:41 2008'' |
Revision as of 10:56, 29 July 2008
Template:STRUCTURE 1ql6
THE CATALYTIC MECHANISM OF PHOSPHORYLASE KINASE PROBED BY MUTATIONAL STUDIES
Template:ABSTRACT PUBMED 10545198
About this Structure
1QL6 is a Single protein structure of sequence from Oryctolagus cuniculus. Full crystallographic information is available from OCA.
Reference
Catalytic mechanism of phosphorylase kinase probed by mutational studies., Skamnaki VT, Owen DJ, Noble ME, Lowe ED, Lowe G, Oikonomakos NG, Johnson LN, Biochemistry. 1999 Nov 2;38(44):14718-30. PMID:10545198
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