Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

Abstract

Human thrombin utilizes Na؉ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na ؉ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na ؉ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na ؉ . This property makes the murine enzyme more resistant to Thrombin is a Na ϩ -activated serine protease (1) that is responsible for the progression and regulation of blood coagulation (2). As in other monovalent cation-activated enzymes (3), the role of Na ϩ in thrombin function is to lower energy barriers for substrate binding in the ground and transition states so that physiologic substrates like PAR1 and fibrinogen can be hydrolyzed efficiently during platelet activation and formation of a blood clot. Other members of the vitamin K-dependent family of clotting proteases to which thrombin belongs are endowed with Na ϩ activation (4). In fact, the activity of activated protein C (5) and coagulation factors VIIa (6), IXa (7), and Xa (8) is influenced significantly by the presence of Na ϩ .Details of the molecular mechanism of Na ϩ activation in thrombin have emerged recently from mutagenesis and structural analysis (9). Na ϩ binding is severely compromised (Ͼ30-fold increase in K d ) upon mutation of Asp-189, Glu-217, Asp-222, and Tyr-225. Asp-189 fixes the orientation of one of the four water molecules ligating Na ϩ and provides an important link between the Na ϩ site and the P1 residue of substrate (10). Glu-217 makes polar contacts with Lys-224 and Thr-172, which helps to stabilize the intervening 220-loop in the Na ϩ site. The ion pair between Arg-187 and Asp-222 latches the 186-loop onto the 220-loop to stabilize the Na ϩ site and the pore of entry of the cation to its binding site (11). Tyr-225 plays a crucial role in determining the Na ϩ -dependent allosteric nature of serine proteases (4) by allowing the correct orientation of the backbone oxygen of residue 224 (12), which contributes directly to the coordination of Na ϩ . The side chain of Tyr-225 also secures the integrity of the water channel embedding the primary specificity pocket (12), and the backbone around Tyr-225 is oriented like the selectivity filter of the KcsA K ϩ channel (3, 13). Of these four residues, Glu-217 and Tyr-225 are conserved in thrombin from all species sequenced to date, from hagfish to human (14). Asp-189 is a Ser in the sturgeon, and the D189S mutant of human thrombin has impaired Na ϩ binding and substrate recognition (10). Asp-222 is the least conserved residue among the four. It is Ser in the sturgeon, Lys in the mouse, and Asn in the rat (14). The substitution in the mouse is particularly interesting, as it involves a charge reversal in the 220-loop that has the potential to destabilize the Na ϩ binding environment.Does the presence of Lys-222 in murine thrombin preclude Na However, the mutant has functional properties interm...

Important Role of the Cys-191–Cys-220 Disulfide Bond in Thrombin Function and Allostery

“…Activity toward this substrate is also compromised due to increased K m and reduced binding but without the increase in k cat observed for FPR. Finally, the chymotrypsin-specific substrate FPF, for which thrombin shows considerable activity unlike trypsin (27), experiences only a modest drop in k cat /K m in the slow form and a more pronounced drop in the fast form. The limited solubility of this substrate prevents independent assessment of the indi- vidual Michaelis-Menten parameters.…”

Structural identification of the pathway of long-range communication in an allosteric enzyme

“…If E* is indeed an inactive conformation of thrombin (47,52), then the recent structure of the mutant D102N in the free form offers a possible representation (42). Given the current structural assignments of E*, E, and E:Na ϩ , one can conclude that the structural changes in the 60-loop documented upon PAR3 binding to exosite I of murine thrombin (32) pertain to a conformation of the enzyme that is stabilized in the highly active E:Na ϩ form (53,54). In fact, murine thrombin is constitutively stabilized in the E:Na ϩ form by the D222K replacement in the Na ϩ site, where Lys-222 provides molecular mimicry of the bound Na ϩ (54).…”

Is it possible to transform trypsin to thrombin?

“…Construction of a thrombin-like protease could focus on either the pro-or anti-coagulant state of the enzyme, yet must rely on the allosteric network responsible for Na þ activation. Recent studies on murine thrombin have demonstrated that thrombin may be locked into the Na þ -bound state by using the Nz atom of K222 as an internal monovalent cation (18). Further engineering of exosite I would be necessary to introduce the fibrinogen recognition site.…”