Sg. Wang et al., Zymogen activation in the streptokinase-plasminogen complex - Ile1 is required for the formation of a functional active site, EUR J BIOCH, 267(13), 2000, pp. 3994-4001
Plasminogen (Plgn) is usually activated by proteolysis of the Arg561-Val562
bond. The amino group of Val562 forms a salt-bridge with Asp740, which tri
ggers a conformational change producing the active protease plasmin (Pm). I
n contrast, streptokinase (SK) binds to Plgn to produce an initial inactive
complex (SK.Plgn) which subsequently rearranges to an active complex (SK.P
lgn*) although the Arg561-Val562 bond remains intact. Therefore another res
idue must substitute for the amino group of Val562 and provide a counterion
for Asp740 in this active complex. Two candidates for this counterion have
been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investig
ated the reaction of SK mutants and variants of the protease domain of micr
oplasminogen (mu Plgn) in order to determine if either of these residues is
the counterion. The mutation of Ile1 of SK decreases the activity of SK.Pl
gn* by 100-fold (Ile1Val) to greater than or equal to 10(4)-fold (Ile1 -->
Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity
of SK, which suggests that Ile1 is not required for formation of SK.Plgn b
ut is necessary for SK.Plgn*. The substitution of Lys698 of mu Plgn decreas
es the activity of SK.Plgn* by only 10-60-fold. In contrast with the Ile1 s
ubstitutions, the Lys698 mutations also decreased the dissociation constant
of the SK complex by 15-50-fold. These observations suggest that Lys698 is
involved in formation of the initial SK.Plgn complex. These results suppor
t the hypothesis that Ile1 provides the counterion for Asp740.