Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.
Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.
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Department of Biochemistry and Molecular Biology, University of North Texas, Fort Worth 76107.
Yüksel
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D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D002645:Chickens; D010450:Endopeptidases; D004795:Enzyme Stability; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry; D011817:Rabbits; D013329:Structure-Activity Relationship; D013552:Swine; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000187:drug effects
D000577:Amides; D009943:Organophosphorus Compounds; C100168:chloroacetol phosphate; D010450:Endopeptidases; D014305:Triose-Phosphate Isomerase