Effect of active-site modification on the terminal marking deamidation of triosephosphate isomerase.
The conformational change which results from the opening and closing of the hinged lid over the catalytic center of triosephosphate isomerase is transmitted to the subunit interface of the dimer and eventually leads to the spontaneous, specific deamidation of Asn71. This is followed by the deamidation of Asn15 approximately 5 A away on the neighboring subunit and leads to destabilization of the protein and degradation. However, it has not been established whether this molecular wear and tear occurs via an intra-subunit or inter-subunit transmission of the conformational change. We have studied this first step in the terminal marking by reacting the active-site Glu165 with the substrate analog 3-chloroacetol phosphate (CAP) which immobilizes the hinged lid. Under mild deamidation conditions (pH 9.5, 24 h, 30 degrees C) the native homodimer readily deamidated. In contrast, the CAP-modified homodimers were resistant to deamidation. Heterodimers composed of one native subunit and one CAP subunit showed intermediate deamidation. When the native and CAP-labeled subunits were resolved by electrophoresis in urea gels, it was found that the unlabeled subunit had preferentially deamidated. These data coupled with molecular modeling considerations show that restricting movement of the hinged lid prevents deamidation of Asn71 on that subunit, but that the other subunit with the free hinged lid functions independently and still deamidates. Thus, the conformationally induced wear and tear appears to be largely intra-subunit. These observations clearly link catalysis with terminal marking of the protein and suggest a general mechanism for how other enzymes may wear out.
Talent
J M
JM
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107, USA.
Zvaigzne
A I
AI
Agrawal
N
N
Gracy
R W
RW
D000577:Amides; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009943:Organophosphorus Compounds / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*
D000577:Amides; D046911:Macromolecular Substances; D009943:Organophosphorus Compounds; C100168:chloroacetol phosphate; D001216:Asparagine; D014305:Triose-Phosphate Isomerase