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We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms that control the balance between the plasminogen activating and plasmin(ogen) binding capacities of H46A, we describe here its gapC gene encoding 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) To study the properties of the GapC protein, its gene was inducibly overexpressed in Escherichiu cnli from QIAexpress expression plasmids to yield the authentic GapC or (His),GapC carrying a hexahistidyl N-terminus to permit affinity purification. Both proteins were functionally active, exhibiting specific GraP-DH activities of about 80 kat/mol ( 4 3 0 Uimg) after purification. Their binding parameters [association (k,,) and dissociation (kc,) rate constants, and equilibrium dissociation constants (KCI = k&J] for the interaction with human Gluplasminogen and plasmin were determined by real-time biospecific interaction analysis using the Pharmacia BIAcore instrument. For comparative purposes, the commercial GraP-DH from Bacillus steurothermuphilus (RstGraP-DH), a nonpathogenic organism, was included in these experiments. The K,, values for binding of plasininogen to GapC, (His),GapC and BstGraP-DH were 220 nM, 260 nM and 520 nM, respectively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the binding to plasmin. These data show that both the zymogen and active enzyme posse low-affinity binding sites for the gupC gene product and that the hexahistidyl terminus does not affect function. Prior limited treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP-DHs, presumably by the exposure of new C-terminal lysine residues for binding to the zymogen.Keywcwds: Streptococcus equisimilis; glyceraldehyde-3-phosphate dehydrogenase; gapC gene; plasmin-(ogen)-binding protein.As a major surprise of recent investigations into the nature of cell surface proteins of the pathogenic streptococci, a wallassociated plasmin binding protein has been discovered that structurally and functionally belongs to the glyceraldehyde-3- Ahhrc.iYntions. BHI, brain heart infusion ; GraP-DH, generic abbreviation [or glyceraldehyde-3-phosphate dehydrogenases: BstGraP-DH, GraP-DH from B~d r ' i i s .stearotlirrmr~philu.~ ; IPTG, isopropyl p-u-thiogalactopyranoside; LBS, lysine-binding site; Ni-NTA, Ni2--nitrilotriacetic acid; ~-~~l-Leii-Lys-CH,Cl, i~-valyl-leucyl-lysyI-chloromethanethane.En~rtzes. Glycelaldel?yde-3-phosphate dehydrogenase (EC 1.2.1.12); plasmin (EC 3.4.21.7).Note. The novel nucleotide sequence data published here has been deposited with the EMBLIGenBanWDDBJ sequence data banks and is available under accesibion number X97788. Broder et al. (1991) were the first to purify from the surface of a group A stre...

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SummaryStructure/function relationships in the activation of plasminogen with staphylokinase were studied using mutants of recombinant staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42DΔN10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,KllH) and Sak42D(K6H,K8H,KllH) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42Δ(K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42DΔCl, or of Lysl35 and Lysl36 in Sak42DΔC2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 × 105 M−1 and 1.4 × 107 M−1, respectively (as compared to a Ka of 1.1 × 108 M−1 for Sak42D).These results indicate that Lysl 1 and the COOH-terminal region of staphylokinase play a key role in the activation of plasminogen.

Three natural variants (wild-type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43Hlstaphylokinase) of the bacterial plasminogen-activator staphylokinase, a 136-aminoacid protein secreted by certain Staphylococcus aureus strains, have been characterized. These variants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogenactivating capacity and conformation in solution, as revealed by fluorescence spectroscopy, dynamic light scattering and circular dichroism. However, the thermostability of these variants is significantly different.At 70°C and 0. ]staphylokinase, respectively. Dynamic light-scattering measurements indicated that inactivation was associated with protein aggregation, which precluded accurate determination of transition temperatures and enthalpies of unfolding. 0.08 -0.34 mg/ml [G34S, R36G, R43H]staphylokinase, however, did not aggregate at 70°C but underwent unfolding as revealed by a 20% increase in the Stokes' radius and a 30% decrease in circular dichroism. The unfolding was reversible upon cooling and was associated with full recovery of functional activity.Thus, these natural variants of staphylokinase have a different sensitivity to thermal inactivation, that is mediated by reversible unfolding of the protein and concentration-dependent irreversible aggregation. [G34S, R36G, R43H]staphylokinase, the most resistant natural variant, has a stability approaching the minimal requirements for pasteurization, which would facilitate its development for clinical use.The thermostability of proteins is an important property for potential industrial or medical applications. The parameters determining thermostability are not well defined and approaches to increase the stability of proteins are lacking [l]. The structural basis of the thermostability of proteins with known three-dimensional structure may be investigated either by specific amino acid replacements via site-directed mutagenesis [2-61, or by analysis of spontaneous or induced genetic variants [7, 81. Staphylokinase, a 136 amino acid protein secreted by certain Staphylococcus aureus strains after lysogenic conversion by phages of different serological groups, activates the plasma fibrinolytic system of several species [9]. Three genes Abbreviations. Wild-type staphylokinase, staphylokinase with glycine, arginine and arginine residues at positions 34, 36 and 43, respectively ; [R36G, R43H]staphylokinase, variant of wild-type staphylokinase with Arg36 and Arg43 replaced by glycine and histidine, respectively; [G34S, R36G, R43H]staphylokinase, variant of wild-type staphylokinase with Arg36 and Arg43 replaced by glycine and histidine, respectively, and Gly34 replaced by serine; AH,, enthalpy of unfolding ; t,, transition temperature encoding wild-type variants of staphylokinase (sak@C, sak42D, sakSTAR) have been characterized [lo -121 which differ at four nucleotide positions within the staphylokinase coding region. Three of these differences represent amino acid exchanges, corresponding to positi...