| Involvement of tyrosine-76 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability and its omega-amino acid ligand binding site. | |
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MedLine Citation:
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PMID: 8142348 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A series of conservative and radical mutations have been made at an aromatic residue, Y76, of the isolated kringle 2 domain of tissue-type plasminogen activator ([K2tPA]) in order to assess the importance of this residue in the ligand binding properties and structural stability of this protein domain. We have successfully expressed in Escherichia coli r-[K2tPA] variants with the following amino acid mutations at Y76: Y76-->A, Y76-->E, Y76-->F, Y76-->K, Y76-->L, Y76-->Q, and Y76-->W. The binding constants of 6-aminohexanoic acid (EACA) and 7-aminoheptanoic acid (7-AHpA) to each of these mutants were investigated by titration of the alterations in intrinsic fluorescence of the mutant kringles with these amino acid ligands. Compared to the wild-type kringle (r-[K2tPA]), which possessed dissociation constants (Kd) of 43 and 6 microM, respectively, for EACA and 7-AHpA, only the Y76-->E mutant displayed a substantially increased Kd value for these amino acids, viz., 117 microM for 7-AHpA. More moderate increases in this parameter were observed for the Y76-->A and Y76-->K variants (2-3-fold increases in the Kd), with no significant differences noted in the cases of Y76-->L, Y76-->Q, and Y76-->W. A most interesting observation was made with the Y76-->F mutant, which showed a 4-6-fold reduction in the Kd for these amino acid ligands. The conformations of all of the mutants were less stable than that of wtr-[K2tPA], as revealed by thermal denaturation studies, suggesting that a Y at sequence position 76 is of importance to the conformational stability of this kringle domain.(ABSTRACT TRUNCATED AT 250 WORDS) |
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Authors:
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V S De Serrano; F J Castellino |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Biochemistry Volume: 33 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 1994 Mar |
Date Detail:
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Created Date: 1994-05-02 Completed Date: 1994-05-02 Revised Date: 2008-11-21 |
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: 3509-14 Citation Subset: IM |
Affiliation:
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Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Amino Acids / metabolism* Base Sequence Binding Sites Crystallography, X-Ray Drug Stability Escherichia coli / genetics Hot Temperature* Kringles* Magnetic Resonance Spectroscopy Models, Molecular Molecular Sequence Data Mutagenesis Protein Conformation Protein Denaturation Recombinant Proteins / chemistry Tissue Plasminogen Activator / chemistry*, genetics Tyrosine* |
| Grant Support | |
ID/Acronym/Agency:
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HL-13423/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Recombinant Proteins; 55520-40-6/Tyrosine; EC 3.4.21.68/Tissue Plasminogen Activator |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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