E. coli D-glyceraldehyde-3-phosphate dehydrogenase modified by 2,3-butanedione: manifestation of a pairwise of non-equivalence of active centers.
Chemical modification of E. coli d-glyceraldehyde-3-phosphate dehydrogenase by an arginine-specific reagent, 2,3-butanedione, stabilized the tetrametric enzyme in an asymmetric state, with only two of the four active centers able to catalyze oxidative phosphorylation of D-glyceraldehyde-3-phosphate. The catalytically incompetent active centers retain the capacity of binding NAD+, forming charge transfer complex, and be alkylated by iodoacetamide. Analogous results have been previously obtained with the rabbit muscle D-glyceraldehyde dehydrogenase modified at a single arginine residue per subunit (Kuzminskaya, E.V., Asryants, R.A., and Nagradova, N.K. (1991) Biochim. Biophys. Acta 1075, 123-130), the only differences being inaccessibility of the catalytically incompetent pair of active centers to the alkylating reagent, on one hand, and lower residual activity exhibited by the functioning active centers (3-4%), on the other. In the case of E. coli enzyme, activity loss upon arginine modification never exceeded 80-82%. These results are consistent with the idea that the two enzymes share common principles of the protein design, but differ in the peculiarities of their active centers conformations. An improved method for D-glyceraldehyde-3-phosphate dehydrogenase purification from a wild type E. coli strain is described.
Levashov
P A
PA
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Schmalhausen
E V
EV
Muronetz
V I
VI
Nagradova
N K
NK
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004852:Epoxy Compounds / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits
D004852:Epoxy Compounds; C007366:diepoxybutane; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases