CLONING, SEQUENCING AND FUNCTIONAL OVEREXPRESSION OF THE STEPTOCOCCUS-EQUISIMILIS H46A GAPC GENE ENCODING A GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE THAT ALSO FUNCTIONS AS A PLASMIN(OGEN)-BINDING PROTEIN-PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE PROTEIN

We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gent: from Streptococcus equisi milis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms th at control the balance between the plasminogen activating and plasmin( ogen) binding capacities of H46A, we describe here its gapC gene encod ing glyceraldehyde-3-phosphate dehydrogenase (GraP-DH, EC 1.2.1.12), a glycolytic enzyme apparently transported to the cell surface where it functions as a plasmin(ogen) binding protein. The gapC gene was clone d and sequenced and found to code for a 336-amino-acid polypeptide (ap proximate to 35.9 kDa) exhibiting 94.9% sequence identity to the Plr p rotein from Streptococcus pyogenes shown by others to be capable of pl asmin binding [Lottenberg, R., Broder, C. C., Boyle, M. D., Kain, S. J ., Schroeder. B. L. & Curtiss, R. III (1992) J. Bacteriol. 174, 5204-5 210]. To study the properties of the GapC protein, its gene was induci bly overexpressed in Escherichia coli from QIAexpress expression plasm ids to yield the authentic GapC or (His)(6)GapC carrying a hexahistidy l N-terminus to permit affinity purification. Both proteins were funct ionally active, exhibiting specific GraP-DH activities of about 80 kat /mol (approximate to 130 U/mg) after purification. Their binding param eters [association (k(n)) and dissociation (k(d)) rate constants, and equilibrium dissociation constants (K-d = k(d)/k(a))] for the interact ion with human Glu-plasminogen and plasmin were determined by real-tim e biospecific interaction analysis using the Pharmacia BIAcore instrum ent. For comparative purposes, the commercial GraP-DH from Bacillus st earothermophilus (BstGraP-DH), a nonpathogenic organism, was included in these experiments. The K-d values for binding of plasminogen to Gap C, (His)(6)GapC and BstGraP-DH were 220 nM, 260 nM and 520 nM, respect ively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the bi nding to plasmin. These data show that both the zymogen and active enz yme possess low-affinity binding sites for the gapC gene product and t hat the hexahistidyl terminus does not affect its function. Prior limi ted treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP-DHs, presumably by the exposure of new C-t erminal lysine residues for binding to the zymogen.