Human erythrocyte bisphosphoglycerate mutase: inactivation by glycation in vivo and in vitro.
2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. 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 study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino 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 were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.
Fujita
T
T
Department of Biochemistry, Osaka University Medical School, Suita, Osaka, 565-0871, Japan.
Suzuki
K
K
Tada
T
T
Yoshihara
Y
Y
Hamaoka
R
R
Uchida
K
K
Matuo
Y
Y
Sasaki
T
T
Hanafusa
T
T
Taniguchi
N
N
D000368:Aged; D000595:Amino Acid Sequence; D001665:Binding Sites; D001731:Bisphosphoglycerate Mutase / Q000302:isolation & purification / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003920:Diabetes Mellitus / Q000201:enzymology*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D006031:Glycosylation; D006801:Humans; D008239:Lysine; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D001731:Bisphosphoglycerate Mutase; D008239:Lysine