Protease activity in single-chain prekallikrein.

Area of Science:

• Biochemistry
• Protease activity
• Contact activation pathway

Background:

• Prekallikrein (PK) is a precursor to kallikrein (PKa), involved in releasing bradykinin and activating factor XII (FXII).
• Reciprocal activation of PK and FXII to their active forms (PKa and FXIIa) is surface-mediated (contact activation).
• FXII shows low intrinsic proteolytic activity, potentially initiating its own activation with PK.

Purpose of the Study:

• To investigate whether prekallikrein (PK) possesses similar intrinsic proteolytic activity.
• To determine if PK can initiate reciprocal activation with FXII.

Main Methods:

• Prepared recombinant PK-R371A, a single-chain mutant unable to convert to PKa.
• Assessed PK-R371A's ability to cleave high-molecular-weight kininogen (HK).
• Evaluated PK-R371A's capacity to convert FXII to FXIIa on a surface.

Main Results:

• PK-R371A cleaved HK, releasing bradykinin with significantly lower catalytic efficiency than PKa.
• PK-R371A converted FXII to FXIIa on a surface, albeit with much lower specific activity than PKa.
• Intrinsic activity of PK and FXII can initiate their reciprocal activation.

Conclusions:

• PK, like FXII, possesses intrinsic proteolytic activity in its precursor form.
• This intrinsic activity can initiate the contact activation pathway.
• The findings support the hypothesis that zymogens of trypsin-like proteases are active and can initiate enzyme cascades.