Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin

Steven J Orcutt,Sriram Krishnaswamy

doi:10.1074/jbc.m410866200

Abstract

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.

Highlights

The formation of thrombin, a key reaction of the blood coagulation cascade, arises from specific and limited proteolysis of prothrombin [1]
We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (IIWT) contained both cleavage sites; IIQ271 contained a single cleavable site at Arg320; IIQ320 and IIA320 contained a single cleavable site at Arg271; and IIQQ was resistant to cleavage
Our results are consistent with a model that accounts for prothrombin activation by prothrombinase based on the measured kinetic properties of each of the four possible cleavage reactions with mutually exclusive binding interactions between each of the four possible substrate species and the enzyme (Scheme II)

Summary

Introduction

The formation of thrombin, a key reaction of the blood coagulation cascade, arises from specific and limited proteolysis of prothrombin [1]. Thrombin formation results from cleavage of human prothrombin following Arg271 and Arg320 [5, 6]. 4) yields thrombin via the formation of prethrombin 2 and fragment 1.2 (P2 plus F1.2) 2 as intermediates This cleavage pathway is evident in the action of factor Xa on prothrombin [7, 8]. Cleavage at Arg320 followed by cleavage at Arg271 (Scheme I, Reactions 1 and 2) results in thrombin formation via production of meizothrombin (mIIa) as an intermediate. Bond cleavage in this order appears to quantitatively account for thrombin formation catalyzed by prothrombinase (9 –11). An explanation for ordered bond cleavage by prothrombinase requires that Arg271 and Arg320 in intact prothrombin are cleaved with different catalytic efficiencies

Methods

Results

Conclusion