Plasmin reduction by phosphoglycerate kinase is a thiol-independent process.
Phosphoglycerate kinase (PGK) is secreted by tumor cells and facilitates reduction of disulfide bond(s) in plasmin (Lay, A. J., Jiang, X.-M., Kisker, O., Flynn, E., Underwood, A., Condron, R., and Hogg, P. J. (2000) Nature 408, 869-873). The angiogenesis inhibitor, angiostatin, is cleaved from the reduced plasmin by a combination of serine- and metalloproteinases. The chemistry of protein reductants is typically mediated by a pair of closely spaced Cys residues. There are seven Cys in human PGK, and mutation of all seven to Ala did not appreciably affect plasmin reductase activity, although some of the mutations perturbed the tertiary structure of the protein. Cys-379 and Cys-380 are close to the hinge that links the N- and C-terminal domains of PGK. Alkylation/oxidation of Cys-379 and -380 by four different thiol-reactive compounds reduced plasmin reductase activity to 7--35% of control. Binding of 3-phosphoglycerate and/or MgATP to the N- and C-terminal domains of PGK, respectively, triggers a hinge bending conformational change in the enzyme. Incubation of PGK with 3-phosphoglycerate and/or MgATP ablated plasmin reductase activity, with half-maximal inhibitory effects at approximately 1 mm concentration. In summary, reduction of plasmin by PGK is a thiol-independent process, although either alkylation/oxidation of the fast-reacting Cys near the hinge or hinge bending conformational change in PGK perturbs plasmin reduction by PGK, perhaps by obstructing the interaction of plasmin with PGK or perturbing conformational changes in PGK required for plasmin reduction.
Lay
Angelina J
AJ
Centre for Thrombosis and Vascular Research, School of Medical Sciences, University of New South Wales, Australia.
Jiang
Xing-Mai
XM
Daly
Elise
E
Sun
Lisa
L
Hogg
Philip J
PJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000478:Alkylation; D005341:Fibrinolysin / Q000378:metabolism*; D005988:Glyceric Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000502:physiology*; D013696:Temperature
D005988:Glyceric Acids; D007527:Isoenzymes; D013438:Sulfhydryl Compounds; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; C104317:phosphoglycerate kinase, testis specific; D005341:Fibrinolysin