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

• Published in: Biochemistry (Easton) Vol. 33; no. 12; pp. 3509 - 3514
• Main Authors: De Serrano, Vesna S, Castellino, Francis J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 29-03-1994
• Subjects: Amino Acid Sequence; Amino Acids - metabolism; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Crystallography, X-Ray; Drug Stability; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hot Temperature; Hydrolases; Kringles; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutagenesis; Protein Conformation; Protein Denaturation; Recombinant Proteins - chemistry; Tissue Plasminogen Activator - chemistry; Tissue Plasminogen Activator - genetics; Tyrosine; Tyrosine; kringle Domain; Ligand binding; Serine endopeptidases; Enzyme; Site directed mutagenesis; Hydrolases; Binding site; Proteinases; Structure function relationship; Thermal stability; t-Plasminogen activator
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00178a007

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.