Probing the catalytic sites of triosephosphate isomerase by ³¹P‐NMR with reversibly and irreversibly binding substrate analogues

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-TPQ2 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.Triosephosphate isomerase (TPI) has been the topic of extensive structural and mechanistic studies for over two decades. The enzyme has emerged as a near-perfect catalyst [l] and the prototype of an eight-stranded alp-barrel structure common in enzyme topology [2]. This conservative, housekeeping enzyme has also provided a model system for the study of postsynthetic modifications of proteins and the initial steps involved in protein degradation [3].TPI is a dimer of identical subunits, each with a catalytic center, and an estimated association constant of l O I 4 -IOl6 M- ' [4]. There is no evidence for cooperativity between the two subunits, but only the dimer possesses isomerase activity [ 5 ] . The two active centers and the subunit interfaces are in close contact via an interdigitating loop that extends from one subunit to the other near the active-site pockets. Gl...

Derivatization of the Interface Cysteine of Triosephosphate Isomerase from Trypanosoma brucei and Trypanosoma cruzi as Probe of the Interrelationship between the Catalytic Sites and the Dimer Interface

“…Loop 3 of one subunit projects between loops 1 and 4 of the other subunit; in this The slopes were then plotted against DTNB or MMTS concentrations, and the apparent rate constants ((, standard error) for the reaction were calculated from these slopes. arrangement, the side chain of cysteine 14 (Figure 4), which is part of loop 1 (residues [14][15][16][17][18][19], becomes closely packed within the core of loop 3 (residues 66-79). Indeed, in the two monomers of TcTIM, there are 12 and 13 atoms at a distance of less than 4 Å from the sulfur of the interface cysteine 14 (Table 3).…”

An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia

“…This suggests that the tetramer is an enzyme with only two catalytically competent sites. In this connection, it is relevant to point out that TIM dimers, in which one of its two catalytic sites has been poisoned with a covalently linked inhibitor, express 50% of its maximal activity without important changes in Km,45, 46 indicating that the catalytic sites of two monomers work independently. However, Biemann and Koshland47 reported that a protein with two potential binding sites exhibited Michaelis–Menten behavior with a Hill coefficient of 1 and that, nonetheless, the protein expressed what they called half of the site reactivity.…”

How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics

“…Although each identical subunit carries an independent catalytic site, TIM is active in the dimeric form (except in some hyperthermophilic bacteria, where its functional form is tetrameric). However, cooperativity or allostery has not been observed between the two active sites (1). Each subunit (with 251 residues in TcTIM) adopts the TIM-barrel topology, in which the active site is located at the C-terminal end of the b-barrel.…”