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  • Title: Probing the catalytic sites of triosephosphate isomerase by 31P-NMR with reversibly and irreversibly binding substrate analogues.
    Author: Schnackerz KD, Gracy RW.
    Journal: Eur J Biochem; 1991 Jul 01; 199(1):231-8. PubMed ID: 2065677.
    Abstract:
    We have explored the degree of independence of the two catalytic centers, interactions between the catalytic centers and the subunit-subunit contact sites, and different conformations of triosephosphate isomerase (TPI), by simultaneously employing irreversibly (covalent) and reversibly binding substrate analogues and monitoring their 31P-NMR resonances. 3-Chloroacetol phosphate (CAP) was bound to the active site by reaction with Glu165. The resulting, inactive (CAP-TPI)2 complex exhibited two distinct 31P-NMR resonances which were independent of pH and represent two conformational forms of the enzyme. Dissociation in guanidine hydrochloride followed by redimerization resulted in a single conformation. This was observed with the enzyme from chicken, rabbit and yeast. The inactive (CAP-TPI)2 dimer was mixed with native TPI, and dissociated/reassociated to form heterodimers (CAP-TPI)(TPI) in which one subunit contained the CAP label and the other subunit was unmodified. This hybrid migrated intermediate between the native and CAP-modified enzyme on nondenaturing PAGE. The heterodimer exhibited 50% the activity of the native dimer, but kinetic properties were otherwise indistinguishable. The reversibly binding transition state analogue, 2-phosphoglycolate (PGA), was used to probe the remaining vacant active site of the heterodimer. Bound PGA exhibited a pH-independent 31P-NMR resonance which was readily distinguishable from resonances of CAP-TPI and free PGA. No differences were observed in the binding of PGA to the vacant subunit of the heterodimer or the native dimer, further pointing to the independent nature of the two catalytic centers. However, the (CAP-TPI)(TPI) heterodimer was more susceptible to subunit dissociation in guanidine hydrochloride than the native dimer. Thus, it appears that the two active sites function completely independently of each other, but that the binding of CAP at the active center loosens the subunit-subunit contact. In addition, the two forms of the enzyme-inhibitor complex trapped by reaction with CAP may represent conformations with the hinged lid or flexible loop (residues 166-176) in the open and closed positions.
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