Zymogen activation in the streptokinase-plasminogen complex - Ile1 is required for the formation of a functional active site

Citation
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
Citations number
40
Categorie Soggetti
Biochemistry & Biophysics
Journal title
EUROPEAN JOURNAL OF BIOCHEMISTRY
ISSN journal
00142956 → ACNP
Volume
267
Issue
13
Year of publication
2000
Pages
3994 - 4001
Database
ISI
SICI code
0014-2956(200007)267:13<3994:ZAITSC>2.0.ZU;2-6
Abstract
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.