CALCIUM-ION MODULATION OF MEIZOTHROMBIN AUTOLYSIS AT ARG(55)-ASP(56) AND CATALYTIC ACTIVITY

When a recombinant variant of prothrombin with the cleavage site mutat ions 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+ (Cote, H. C, F., Stevens, W. K., Ba jzar, L., Banfield, D. K., Nesheim, M. E., and MacGillivray, R. T. A. (1994) J. Biol. Chem. 269, 11374-11380). In the absence of Ca2+ howeve r, rMZa is rapidly cleaved within a disulfide bonded loop in the Fl do main at Arg(55) in the sequence RTPR down arrow DKL, yielding a molecu le with 3 chains joined by two disulfide bonds (rMZa). Cleavage kinet ics are first order regardless of the rMZa concentration, indicating a n intramolecular cleavage. This cleavage does not occur at Ca2+ concen trations in excess of 1.0 mM. To assess the role of the F1 domain in r MZa activity, another variant lacking the R155A mutation (rMZdesF1) wa s expressed, which when activated yields meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of the tripeptide substrate S2 238 by rMZa or rMZa increase from 60% to 90% that of recombinant thro mbin as Ca2+, Mg2+, or Mn2+ concentrations are varied from 0 to 10 mM. K-m and k(cat) values for rMZa in the absence and presence of 5 mM Ca 2+ are 1.9 and 2.2 mu M and 65 and 105 s(-1), TAME esterase activity o f rMZa also increases with 5 mM Ca2+, No such metal ion-dependent effe cts are obtained with either thrombin or rMZdesF1a. Fibrinogen clottin g activities, relative to that of thrombin, increase in a manner analo gous to those obtained with small substrates, for rMZa and rMZa but n ot rMZdesF1a, Complexes of the active site probe dansylarginine N-(3-e thyl-1,5-pentanediyl)amide with rMZa and rMZa, but not thrombin or rM ZdesF1a exhibit large cation-dependent decreases in fluorescence inten sity, suggesting that metal ion binding in the F1 domain alters the en vironment 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 th e F1 domain but also the protease domain.