Ratcheting between Two Distinct Conformations of Substrate Drives the Sequential Cleavage of Prothrombin by Prothrombinase.
Authors: Bianchini Elsa , Orcutt Steven J. , Krishnaswamy Sriram 
Published: October 2019

Abstract
The conversion of human prothrombin to thrombin requires the cleavage of two peptide bonds. When catalyzed by prothrombinase, the reaction proceeds almost exclusively via initial cleavage at R320 followed by cleavage at R271 yielding meizothrombin (mIIa) as an intermediate. The scissile bonds are expected to be ~36 A apart in the zymogen. Yet, remarkably, evidence indicates that cleavage at these two sites is accomplished by a single type of exosite interaction that tethers the substrate to prothrombinase. The ability of prothrombinase to act on these spatially distinct sites, with such constraints, can be explained by a conformational change in the substrate following initial cleavage at R320. Cleavage at this site leads to internal salt bridge formation and a conformational transition to the proteinase. The role of the zymogen to proteinase transition in substrate cleavage was investigated by the use of a fully-carboxylated recombinant prothrombin derivative (IITAT) with the I-V-E sequence following R320 replaced with T-A-T. Thrombin produced by cleavage of IITAT exhibited ~0.2% of the catalytic activity observed with thrombin produced from wild type recombinant prothrombin (IIWT). IITAT can be cleaved at the R320 site but fails to undergo all subsequent conformational changes required for proteinase formation. SDS-PAGE and quantitative densitometry revealed that the action of prothrombinase on either IIWT or IITAT was consistent with ordered cleavage at R320 followed by cleavage at R271. The rates of consumption of IIWT and IITAT resulting from cleavage at R320 were equivalent. Cleavage at R320 in IITAT by prothrombinase is not detectably affected by the substituted P1′-P3′ sequence. The disappearance of IIWT was accompanied by a transient accumulation in mIIa that decreased to zero within 4 minutes while thrombin accumulated following a short delay. With IITAT, mIIa accumulated to higher levels and persisted for 45 minutes. Thrombin was produced with a lower rate and a longer delay phase. The findings imply a substantial decrease in the rate of the second cleavage reaction at R271 resulting from distant effects of the new P1′-P3′ residues following R320. The thrombin inhibitor, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) had no effect on the cleavage reactions in IIWT. However, increasing concentrations of DAPA as high as 400 μM were found to systematically enhance the rate of thrombin formation from IITAT by correcting defective cleavage at R271. The data are consistent with the interpretation that IITAT can be cleaved normally at R320 but subsequent accessibility and cleavage at R271 is reduced because of a defect in internal salt bridge formation and the conformational change associated with proteinase formation. Rescue of this defect by high concentrations of DAPA likely relates to the stabilization of a proteinase-like conformation in the intermediate produced by cleavage of IITAT at R320. Our findings suggest an important role for the zymogen to proteinase transition in determining the sequential action of prothrombinase on the two sites in prothrombin. We propose that exosite-dependent tethering of two distinct conformations of the substrate to prothrombinase drives the presentation of distantly positioned cleavage sites to the active site of the enzyme and accounts for the sequential cleavage of prothrombin.