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. 2010 Jan 1;285(1):328-38.
doi: 10.1074/jbc.M109.070334. Epub 2009 Oct 26.

Regulated cleavage of prothrombin by prothrombinase: repositioning a cleavage site reveals the unique kinetic behavior of the action of prothrombinase on its compound substrate

Harlan N Bradford  1 , Joseph A Micucci, Sriram Krishnaswamy
Affiliations

Regulated cleavage of prothrombin by prothrombinase: repositioning a cleavage site reveals the unique kinetic behavior of the action of prothrombinase on its compound substrate

Harlan N Bradford et al. J Biol Chem. .

Abstract

Prothrombinase converts prothrombin to thrombin via cleavage at Arg(320) followed by cleavage at Arg(271). Exosite-dependent binding of prothrombin to prothrombinase facilitates active site docking by Arg(320) and initial cleavage at this site. Precise positioning of the Arg(320) site for cleavage is implied by essentially normal cleavage at Arg(320) in recombinant prothrombin variants bearing additional Arg side chains either one or two residues away. However, mutation of Arg(320) to Gln reveals that prothrombinase can cleave prothrombin following Arg side chains shifted by as many as two residues N-terminal to the 320 position at near normal rates. Further repositioning leads to a loss in cleavage at this region with an abrupt shift toward slow cleavage at Arg(271). In contrast, the binding constant for the active site docking step is strongly dependent on the sequence preceding the scissile bond as well as position. Large effects on binding only yield minor changes in rate until the binding constant passes a threshold value. This behavior is expected for a substrate that can engage the enzyme through mutually exclusive active site docking reactions followed by cleavage to yield different products. Cleavage site specificity as well as the ordered action of prothrombinase on its compound substrate is regulated by the thermodynamics of active site engagement of the individual sites as well as competition between alternate cleavage sites for active site docking.

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Figures

SCHEME 1.
SCHEME 1.
Cleavage products of prothrombin. Line diagrams illustrate the two cleavage sites within intact prothrombin (II). Numbering is according to the 579 residues in the mature protein. Cleavage at Arg320 yields meizothrombin (mIIa), whereas a single cleavage at Arg271 yields F12 and P2. The species obtained following cleavage at both sites are F12 and IIa. A key disulfide bond that covalently links two polypeptide chains in IIa and mIIa is illustrated to aid in the interpretation of the results of SDS-PAGE following disulfide bond reduction.
SCHEME 2.
SCHEME 2.
Cleavage site variants of prothrombin. The sequence surrounding the two cleavage sites in prothrombin are listed for the variants used in this study. Vertical arrows denote the position of the scissile bonds in wild type prothrombin. Mutated residues are shown in red, and residues in blue denote the new N-terminal sequence following cleavage required for the expression of proteolytic activity in the product. All variants possessed the same sequence flanking the Arg271 site as the wild type species.
FIGURE 1.
FIGURE 1.
Cleavage of IIWT and IIRR by prothrombinase. Reaction mixtures containing 1.4 μm IIWT (A) or IIRR (B), 30 μm PCPS, 60 μm DAPA, and 30 nm Va in Assay Buffer were initiated by the addition of 0.2 nm Xa. Samples were quenched, analyzed by SDS-PAGE following disulfide bond reduction, stained with Colloidal Blue, and visualized as described under “Experimental Procedures.” Lanes 1–18 correspond to reaction times of 0, 0.33, 0.66, 1.0, 1.33, 1.66, 2, 2.33, 2.66, 3, 4, 6, 8, 12, 20, 30, 45, and 65 min. Identities of the relevant species are indicated in the margin, and two gels, aligned using molecular weight markers, are presented in each panel. The IIaA polypeptide species (Scheme 1) runs off the gel with the dye front in this gel system.
FIGURE 2.
FIGURE 2.
Formation of proteinase products from prothrombin variants. Reaction mixtures contained 1.4 μm IIVariant, 30 μm PCPS, 6 μm DAPA, 30 nm Va, and 0.2 nm Xa. The initial velocity of S2238 hydrolysis was determined on aliquots withdrawn and quenched at the indicated times and converted to the concentration of proteinase formed as described under “Experimental Procedures.” Data obtained with IIWT (●), IIRR (○), IIRGR (▴), or II-1δ (▵) are illustrated. The line was arbitrarily drawn.
FIGURE 3.
FIGURE 3.
Cleavage of the register shift prothrombin variants by prothrombinase. Experimental conditions, reactant concentrations, and reaction times were identical to those presented in the legend for Fig. 1 except that the variants analyzed were different. The stained gels illustrate the fate of IIRGR (A), II-1δ (B), II-2δ (C), II-3δ (D), II-4δ (E), and IIQ320 (F) following the addition of prothrombinase. Identities of the polypeptide species are denoted in the margins.
FIGURE 4.
FIGURE 4.
Fate of prothrombin variants following cleavage by prothrombinase. Progress curves for prothrombin consumption were constructed by quantitative densitometry following fluorescence scanning of gels as illustrated in Figs. 1 and 3 and normalized as described. The data sets correspond to those describing the fate of IIWT (●), IIRR (○), IIRGR (▴), II-1δ (▵), II-2δ (▾), II-3δ (▿), II-4δ (■), and IIQ320 (□). Data points and error bars denote means ± 1 S.D. from 2–5 experiments. The lines were arbitrarily drawn. Initial rates determined from these progress curves are presented in Table 2.
FIGURE 5.
FIGURE 5.
Active site docking by prothrombin variants. Fluorescence measurements at equilibrium were determined using λEX = 320 nm, λEM = 374 nm, and reaction mixtures containing 1 μm XaS195A, 1.2 μm Va, 200 μm PCPS, 10 μm FPR-CH2Cl, 25 μm pAB, and increasing concentrations of the indicated variant. Data are presented as FOBS/FP,Free, reflecting the ratio of measured fluorescence intensity to that for pAB in solution following corrections for scatter and normalization. Titration curves are presented for IIWT (●), IIRR (○), IIRGR (▴), II-1δ (▵), II-2δ (▾), II-3δ (▿), II-4δ (■), and IIQ320 (□). With the exception of II-4δ and IIQ320 for which only one representative experiment is shown, data points and error bars denote means ± 1 S.D. from 3–5 different experiments. The lines are drawn following analysis as described under “Experimental Procedures” using the fitted constants listed in Table 2.
SCHEME 3.
SCHEME 3.
Kinetic pathways for prothrombin binding and cleavage by prothrombinase. The annotated scheme illustrates the initial binding of prothrombin through exosite interactions with prothrombinase determined by KEXO. Exosite-bound substrate then engages the active site through one of two mutually exclusive active site docking steps. Active site engagement by Arg320, determined by Ks*320, leads to the cleavage at the 320 site and the formation of mIIa. Active site engagement by Arg271, determined by Ks*271, leads to the cleavage at the 271 site and the formation of P2 plus F12. Definition of KEXO, Ks*320, and Ks*271 in terms of rate constants is shown, and the intrinsic kcat for cleavage at either the 320 site or the 271 site is listed as kcat320 and kcat271.
FIGURE 6.
FIGURE 6.
Kinetic accounting of the action of prothrombinase on prothrombin variants. Normalized rates ± 2 S.D. for the consumption of all prothrombin variants with the exception of IIQ320 are plotted versus measured values for Ks*320. The solid black line was calculated using Equations 1, 2, and 5 using the values in Table 2 (see “Results”), Ks*271 = 4.3, kcat320 = 94 s−1, and kcat271 = 100 s−1. The red and blue lines denote the fractional contributions of cleavage at the 320 region or the 271 site to the rate of prothrombin cleavage.
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