The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from Trypanosoma brucei: metal-ion dependency and phosphoenzyme formation

Recombinant cofactor-independent phosphoglycerate mutase from Trypanosoma b rucei was inactivated by EDTA, and reactivated by Co2+ much more than by Mn 2+ or Fe2+. It displayed a minor phosphoglycerate phosphatase activity, whi ch was stimulated by Mn2+ more than by Co2+. Upon incubation With [P-32]pho sphoglycerate, radioactivity was incorporated into the enzyme, most particu larly in the presence of Mn2+ or Fe2+. The phosphorylated residue was ident ified by tandem mass spectrometry as Ser74, a residue homologous to the pho sphorylated serine in alkaline phosphatase. However, the rates of formation and of disappearance of this phosphoenzyme were quite low compared to the mutase reaction. This and other properties indicated that the observed phos phoenzyme is an intermediate in the minor phosphatase activity rather than in the phosphomutase reaction. (C) 2001 Federation of European Microbiologi cal Societies. Published by Elsevier Science B.V. All rights reserved.