Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers.
Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.
Zomosa-Signoret
Viviana
V
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70243, 04510 México, D. F., Mexico.
Hernández-Alcántara
Gloria
G
Reyes-Vivas
Horacio
H
Martínez-Martínez
Eduardo
E
Garza-Ramos
Georgina
G
Pérez-Montfort
Ruy
R
Tuena De Gómez-Puyou
Marietta
M
Gómez-Puyou
Armando
A
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D019281:Dimerization; D004789:Enzyme Activation; D007700:Kinetics; D016296:Mutagenesis; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000201:enzymology
D017931:DNA Primers; D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase