Susceptibility to proteolysis of triosephosphate isomerase from two pathogenic parasites: characterization of an enzyme with an intact and a nicked monomer.
The susceptibility to subtilisin of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) was studied. Their amino sequence and 3D structure are markedly similar. In 36 h of incubation at a molar ratio of 4 TIM per subtilisin, TcTIM underwent extensive hydrolysis, loss of activity, and large structural alterations. Under the same conditions, only about 50% of the monomers of TbTIM were cleaved in two sites. The higher sensitivity of TcTIM to subtilisin is probably due to a higher intrinsic flexibility. We isolated and characterized TbTIM that had been exposed to subtilisin. It exhibited the molecular mass of the dimer, albeit it was formed by one intact and one nicked monomer. Its k(cat) with glyceraldehyde 3-phosphate was half that of native TbTIM, with no change in K(m). The intrinsic fluorescence of nicked TbTIM was red-shifted by 5 nm. The association between subunits was not affected. The TbTIM data suggest that there are structural differences in the two monomers or that alterations of one subunit change the characteristics of the other subunit. In comparison to the action of subtilisin on TIMs from other species, the trypanosomal enzymes appear to be unique.
Instituto de Fisiología Celular, Universidad Nacional Autónoma de Mexico, Mexico. hreyes@ifisiol.unam.mx
Horacio
H
Reyes-Vivas
Eduardo
E
Martínez-Martínez
Guillermo
G
Mendoza-Hernández
Gabriel
G
López-Velázquez
Ruy
R
Pérez-Montfort
Marietta
M
Tuena de Gómez-Puyou
Armando
A
Gómez-Puyou
D000595:Amino Acid Sequence; D000818:Animals; D004586:Electrophoresis; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D020860:Subtilisin / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000472:pathogenicity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000472:pathogenicity
D020860:Subtilisin; D014305:Triose-Phosphate Isomerase