| Exploring the role of the active site cysteine in human muscle creatine kinase. | |
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MedLine Citation:
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PMID: 16981706 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized. |
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Authors:
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Pan-Fen Wang; Allen J Flynn; Mor M Naor; Jan H Jensen; Guanglei Cui; Kenneth M Merz; George L Kenyon; Michael J McLeish |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S. |
Journal Detail:
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Title: Biochemistry Volume: 45 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 2006 Sep |
Date Detail:
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Created Date: 2006-09-19 Completed Date: 2006-11-08 Revised Date: 2013-06-07 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 11464-72 Citation Subset: IM |
Affiliation:
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Department of Medicinal Chemistry, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Adenosine Diphosphate
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metabolism Animals Binding Sites Computer Simulation Creatine / metabolism Creatine Kinase, MM Form / chemistry*, metabolism* Cysteine / metabolism* Humans Hydrogen-Ion Concentration Kinetics Models, Molecular Mutant Proteins / chemistry, metabolism Phosphorylation Proline / chemistry Serine / chemistry Spectrophotometry, Ultraviolet Structure-Activity Relationship Torpedo |
| Grant Support | |
ID/Acronym/Agency:
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GM44974/GM/NIGMS NIH HHS; R01 GM044974-16/GM/NIGMS NIH HHS; R01 GM066859-09/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Mutant Proteins; 147-85-3/Proline; 52-90-4/Cysteine; 56-45-1/Serine; 57-00-1/Creatine; 58-64-0/Adenosine Diphosphate; EC 2.7.3.2/Creatine Kinase, MM Form |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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