Calcium ion modulation of meizothrombin autolysis at Arg55-Asp56 and catalytic activity

Abstract

When a recombinant variant of prothrombin with the cleavage site mutations R155A, R271A, and R284A (rMZ) is exposed to either prothrombinase or ecarin, a form of meizothrombin (rMZa) is generated that is stable for weeks in the presence of Ca2+ (Côté, H. C. F., Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., and MacGillivray, R. T. A. (1994) J. Biol. Chem. 269, 11374-11380). In the absence of Ca2+ however, rMZa is rapidly cleaved within a disulfide bonded loop in the F1 domain at Arg55 in the sequence RTPR ↓ DKL, yielding a molecule with 3 chains joined by two disulfide bonds (rMZa*). Cleavage kinetics are first order regardless of the rMZa concentration, indicating an intramolecular cleavage. This cleavage does not occur at Ca2+ concentrations in excess of 1.0 mM. To assess the role of the F1 domain in rMZa activity, another variant lacking the R155A mutation (rMZdesF1) was expressed, which when activated yields meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of the tripeptide substrate S2238 by rMZa or rMZa* increase from 60% to 90% that of recombinant thrombin as Ca2+, Mg2+, or Mn2+ concentrations are varied from O to 10 mM. Km and kcat values for rMZa in the absence and presence of 5 mM Ca2+ are 1.9 and 2.2 μM and 65 and 105 s-1. TAME esterase activity of rMZa also increases with 5 mM Ca2+. No such metal ion-dependent effects are obtained with either thrombin or rMZdesF1a. Fibrinogen clotting activities, relative to that of thrombin, increase in a manner analogous to those obtained with small substrates, for rMZa and rMZa* but not rMZdesF1a. Complexes of the active site probe dansylarginine N-(3-ethyl-1,5-pentanediyl)amide with rMZa and rMZa*, but not thrombin or rMZdesF1a exhibit large cation-dependent decreases in fluorescence intensity, suggesting that metal ion binding in the F1 domain alters the environment of the probe at the active site. These results indicate that in the absence of divalent cations, the activity of rMZa is inhibited, perhaps by obstruction of the active site by the F1 domain, and that Ca2+ binding to the F1 domain modulates the properties of not only the F1 domain but also the protease domain.