Limited proteolysis of triose-phosphate isomerase and characterization of the catalytically active peptide complex.
Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.
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Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107.
Yüksel
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KU
Gracy
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D000595:Amino Acid Sequence; D002384:Catalysis; D004795:Enzyme Stability; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017510:Protein Folding; D012441:Saccharomyces cerevisiae; D013381:Subtilisins / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D013381:Subtilisins; D014305:Triose-Phosphate Isomerase