Human erythrocyte bisphosphoglycerate mutase: Inactivation by glycation invivo and in vitro

2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enz yme that catalyzes both the synthesis and the degradation of 2,3-diphosphog lycerate (2,3-DPG) and contains three types of activities in that it functi ons as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosph atase, In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG, The present stu dy shows that the specific activity of BPGM in erythrocytes of diabetic pat ients is decreased, compared to normal controls as judged by 2,3-DPG synthe tase activity and immunoreactive contents. To understand the mechanism by w hich the enzyme is inactivated, the enzyme was purified from pooled erythro cytes from diabetic patients and subjected to a boronate affinity column. T he dow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an antihexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The p rimary glycated site of the enzyme was found to be Lys158 as judged by amin o acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites w ere Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196, From these results, the l oss of enzymatic activity appears to be due to the glycation of Lys158 whic h may be located in the vicinity of the substrate binding site.