Plasmin Reduction by Phosphoglycerate Kinase Is a Thiol-independent Process

Angelina J Lay,Xing-Mai Jiang,Elise Daly,Lisa Sun,Philip J Hogg

doi:10.1074/jbc.m111531200

Abstract

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.

Highlights

Disulfide bonds of certain cell-surface proteins can interchange between the oxidized and reduced state [1,2,3]
Plasmin is processed in the conditioned medium of tumor cells, producing angiostatin fragments consisting of kringle domains 1– 41⁄2, 1– 4, and 1–3 (Ref. 6 and references therein)
There are seven Cys in Phosphoglycerate kinase (PGK), none of which are involved in disulfide bonds, and only two of the seven are nearby in the primary or tertiary structure [22,23,24,25]

Summary

Introduction

Disulfide bonds of certain cell-surface proteins can interchange between the oxidized and reduced state [1,2,3]. Alkylation/oxidation of Cys-379 and -380 by four different thiol-reactive compounds reduced plasmin reductase activity to 7–35% of control. We show that none of the PGK Cys residues are directly involved in plasmin reduction but that alkylation/oxidation of the fast-reacting Cys or conformational changes in the same region of the protein inhibit reductase activity.

Results

Conclusion