Mutant N143P Reveals How Na+ Activates Thrombin

Abstract

The molecular mechanism of thrombin activation by Na ؉ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na ؉ forms. peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na؉ activation is present in all Na ؉ -activated trypsin-like proteases.Regulation of activity through metal ion complexation plays a key role in many enzyme-catalyzed reactions, and over onethird of known proteins are metalloproteins (1-3). The earliest evidence for monovalent cation (M ϩ ) activation of enzymes was provided by Boyer (4) with the discovery of the absolute requirement of K ϩ by pyruvate kinase (5). Monod demonstrated Na ϩ -dependent catalytic rate enhancement in ␤-galactosidase (6). Following these discoveries, many enzymes were observed to display increased activity in the presence of M ϩ (7). A recent classification of M ϩ

Crystal structure of prethrombin-1

“…An intriguing connection is established with the E Ã -E equilibrium of thrombin (31), meizothrombin (11), factor Xa (32), and activated protein C (32), where E* is an inactive form of the protease with the active site closed that interconverts on the millisecond timescale with the active form E where the active site is accessible to substrate. Indeed, the side chain of W215 in the hydrophobic cluster of prethrombin-1 occupies a position that is very similar to that observed in the E* form of thrombin (12)(13)(14)(15), where it relocates >10 Å from the position assumed in the active E form (33).…”

“…The H bond between residues 143 and 192 is of utmost importance in trypsinlike proteases because it locks the 192-193 peptide bond in an orientation that enables the backbone N atoms of G193 and S195 to point toward the interior of the tight β-turn within the active site that defines the oxyanion hole (23)(24)(25). When the 143-192 H bond is broken, as illustrated directly by the N143P mutant of thrombin (15), the 192-193 peptide bond flips from a type II to a type I β-turn, the backbone O atom of residue 192 points inward toward the active site S195, and the 192-194 segment adopts a 3 10 -helix conformation that disrupts the oxyanion hole. Examples of this perturbed conformation of the oxyanion hole have been reported for the inactive E* form of thrombin (12)(13)(14)(15), complement factor B (26), the arterivirus nsp4 (27), the epidermolyic toxin A (28,29), and clotting factor VIIa (30).…”

“…When the 143-192 H bond is broken, as illustrated directly by the N143P mutant of thrombin (15), the 192-193 peptide bond flips from a type II to a type I β-turn, the backbone O atom of residue 192 points inward toward the active site S195, and the 192-194 segment adopts a 3 10 -helix conformation that disrupts the oxyanion hole. Examples of this perturbed conformation of the oxyanion hole have been reported for the inactive E* form of thrombin (12)(13)(14)(15), complement factor B (26), the arterivirus nsp4 (27), the epidermolyic toxin A (28,29), and clotting factor VIIa (30). Finally, the Oδ atom of N143 engages the backbone N atom of S195 precluding access of substrate to what remains of the oxyanion hole.…”

“…Collapse of the 215-217 segment into the active site and disruption of the oxyanion hole due to a flip of the 192-193 peptide bond are distinctive features of the E* form of thrombin and occur with the I16-D194 ion pair intact (12)(13)(14)(15). A collapsed conformation of the 215-217 segment is also seen in the structures of αI-tryptase (35), the high-temperaturerequirement-like protease (36), complement factor D (37), granzyme K (38), hepatocyte growth factor activator (39), prostate kallikrein (40), and prostasin (41,42).…”

“…1). Here we report the X-ray crystal structure of prethrombin-1 at 2.2-Å resolution that reveals an unprecedented conformation with occlusion of the active site similar to that observed in the inactive E* form of thrombin (11)(12)(13)(14)(15). Several independent lines of evidence suggest that trypsin-like proteases undergo an equilibrium between the inactive E* form and the active E form (5, 6) and that the distribution of these forms dictates the catalytic activity of the enzyme.…”