The cationic locus on the recombinant kringle 2 domain of tissue-type plasminogen activator that stabilizes its interaction with omega-amino acids.
The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-[K2tPA] mutants obtained by oligonucleotide-directed mutagenesis. We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-[K2tPA] and determined the dissociation constants of several omega-amino acids, viz., 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), to each of the r-[K2tPA] variants. We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands. When K33 is altered to a variety of other amino acids, the K33R mutant best stabilizes binding of all of these ligands. However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca. 2-fold reduction in binding strength) as wild-type r-[K2tPA]. Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino acids to the muteins. We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane. These observations show that the omega-amino acid binding site of wtr-[K2tPA] could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
De Serrano
V S
VS
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D015119:Aminocaproic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002412:Cations; D004247:DNA / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014148:Tranexamic Acid / Q000378:metabolism
D000596:Amino Acids; D002412:Cations; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014148:Tranexamic Acid; D004247:DNA; C018819:omega-aminoenantic acid; D010959:Tissue Plasminogen Activator; D008239:Lysine; D015119:Aminocaproic Acid