Reactivation of 3-phosphoglycerate kinase from its unfolded proteolytic fragments.
Limited trypsinolysis of pig muscle 3-phosphoglycerate kinase yielded a nicked enzyme without loss of catalytic activity [Jiang, S. X. & Vas, M. (1988) FEBS Lett. 231, 151-154]. The reactivation rate of the nicked enzyme after denaturation does not differ substantially from the reactivation rate of the denatured intact enzyme: t 1/2 varies between 70-110 s at 25 degrees C, pH 7.0 in both cases. Thus, the absence of a covalent linkage between the two proteolytic fragments of the enzyme molecule apparently does not affect the refolding. The two proteolytic fragments can be separated by FPLC under denaturing conditions. Fluorescence spectra of the isolated fragments may indicate that the tryptic cleavage site is within the N-terminal domain. Thus, the larger fragment (molecular mass about 30 kDa) probably contains the whole nucleotide-binding C-terminal domain plus a small part of the N-terminal domain. The inactive isolated fragments were used in renaturation experiments to study the reassembly of active 3-phosphoglycerate kinase. Kinetic measurements revealed the presence of a bimolecular rate-limiting step of reactivation. Separate preincubation of the fragments under renaturing conditions did not cause substantial acceleration of reactivation. This implies that assembly of the separate structural units (possibly domains) may limit the reactivation of the intact enzyme.
Vas
M
M
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest.
Sinev
M A
MA
Kotova
N V
NV
Semisotnov
G V
GV
D000818:Animals; D002942:Circular Dichroism; D004793:Enzyme Reactivators; D006801:Humans; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism*; D010735:Phosphoglycerate Kinase / Q000032:analysis / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013552:Swine; D014357:Trypsin
D004793:Enzyme Reactivators; D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase; D014357:Trypsin