Energetic and Structural Consequences of Perturbing Gly-193 in theOxyanion Hole of SerineProteases

Abstract

The oxyanion hole of serine proteases is formed by the backbone N atoms of the catalytic Ser-195 and Gly-193 and engages the backbone O atom of the P1 residue of substrate in an important H-bonding interaction. The energetic contribution of this interaction in the ground and transition states is presently unknown. Measurements of the individual rate constants defining the catalytic mechanism of substrate hydrolysis for wild-type thrombin and trypsin and their G193A and G193P mutants reveal that Gly-193 is required for optimal substrate binding and acylation. Crystal structures of the G193A and G193P mutants of thrombin bound to the active site inhibitor H-D-Phe-Pro-Arg-CH 2 Cl document the extent of perturbation induced by the replacement of Gly-193. The Ala mutant weakens the H-bonding interaction of the N atom of residue 193, whereas the Pro substitution abrogates it altogether with additional small shifts of the protein backbone. From the kinetic and structural data, we estimate that the H-bonding interaction in the oxyanion hole contributes a stabilization of the ground and transition states of >1.5 kcal/mol but <3.0 kcal/mol. These results shed light on a basic aspect of the enzyme-substrate interaction in the entire family of trypsin-like serine proteases.Serine proteases catalyze the hydrolysis of peptide bonds using the triad His-57/Asp-102/Ser-195 (chymotrypsinogen numbering) (1, 2). Binding of substrate to the active site is stabilized by a network of H-bonds, five of which are highly conserved and ensure the efficient hydrolysis of amide bonds. Of these H-bonds, two stabilize an anti-parallel ␤-sheet between Gly-216 and the P3 residue of substrate, whereas the other three involve the backbone N and O atoms of the P1 residue of substrate. The N atom engages the backbone O atom of Ser-214, often referred to as the "fourth" member of the catalytic machinery (3). The O atom of the P1 residue, on the other hand, makes two H-bonding interactions with the backbone N atoms of the catalytic in the so-called "oxyanion hole." The role of this region of the enzyme is to stabilize the developing negative charge on the O atom of substrate during formation of the tetrahedral intermediate (4). The lack of a side chain at position 193 allows correct positioning of the amido hydrogen to form the requisite H-bond with the oxyanion substrate. Indeed, Gly is highly conserved at position 193 in serine proteases, but few exceptions do exist and are associated with perturbed substrate hydrolysis and resistance to inhibition (5-8).Previous studies have addressed the important question of the energetic involvement of the oxyanion hole in substrate recognition. In subtlisin, one of the H-bonding group in the oxyanion hole is provided by the side chain of Asn-155 (9). Mutation of Asn-155 to the isosteric Leu, devoid of H-bonding capabilities, produces a derivative with reduced k cat /K m due to a significant (200-fold) decrease of k cat (9, 10). The absence of effect on K m has been interpreted as supporting the role of the H-b...

Mutant N143P Reveals How Na+ Activates Thrombin

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Functional Role of Residue 193 (Chymotrypsin Numbering) in Serine Proteases: Influence of Side Chain Length and β-Branching on the Catalytic Activity of Blood Coagulation Factor XIa

“…This may reflect the energy required to break interactions between the larger side chains of Asp, Val, Glu, and Lys and surrounding residues to allow rotation of the 192-193 bond so that the amide nitrogen of residue 193 points toward the oxyanion hole. In the case of Pro 193 , the residue lacks an amide nitrogen preventing its participation in hydrogen bonding with the oxyanion of the substrate/inhibitor (52). The effects of residue 193 side chains on FXIa activity should apply to proteases such as FVIIa and FIXa, which have similar hydrophobic S2′ sites (36)(37)(38)53,54).…”

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

“…Functional Assays-Wild-type trypsin and tissue-type plasminogen activator were expressed, purified, and tested for activity as described (23,24). Chymotrypsin was from Sigma.…”