Theoretical Perspectives on the Reaction Mechanism of Serine Proteases:  The Reaction Free Energy Profiles of the Acylation Process

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...

Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 74%

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

Bobofchak

Pineda

Mathews

et al. 2005

“…However, for almost all enzymes, a doubledisplacement (ping-pong) kinetic mechanism is described that involves a covalent substrate-enzyme intermediate with the substrate linked to the nucleophile of the catalytic triad (42,58,66,(71)(72)(73)(74)(75)(76). In line with this, a stable acetyl-enzyme intermediate was observed for OatC that crucially depended on the presence of Ser-286.…”

Section: Discussionmentioning

confidence: 85%

The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases

Bergfeld

Claus

Lorenzen

et al. 2009

Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of ␣2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by ␣2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS 286 XGG), which is a hallmark of ␣/␤-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to ␣/␤-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an ␣/␤-hydrolase fold topology, our data provide strong evidence that OatC belongs to the ␣/␤-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed ␤-helix fold and assemble into catalytic trimers.

“…This is not a unique reaction mechanism among proteases. Kinetic evidence based on the acylation of chymotrypsin by specific oxygen and sulfur esters (33) as well as quantum chemical calculations (34) suggest reversible formation of TI 1 . However, these studies reported rate-limiting formation of the tetrahedral intermediate followed by immediate formation of the acyl enzyme.…”

Section: Discussionmentioning

confidence: 99%

Thermodynamic Analysis Reveals Structural Rearrangement during the Acylation Step in Human Trypsin 4 on 4-Methylumbelliferyl 4-Guanidinobenzoate Substrate Analogue

Toth

Gombos

Simon

et al. 2006

Human trypsin 4 is an unconventional serine protease that possesses an arginine at position 193 in place of the highly conserved glycine. Although this single amino acid substitution does not affect steady-state activity on small synthetic substrates, it has dramatic effects on zymogen activation, interaction with canonical inhibitors, and substrate specificity toward macromolecular substrates. To study the effect of a non-glycine residue at position 193 on the mechanism of the individual enzymatic reaction steps, we expressed wild type human trypsin 4 and its R193G mutant. 4-Methylumbelliferyl 4-guanidinobenzoate has been chosen as a substrate analogue, where deacylation is rate-limiting, and transient kinetic methods were used to monitor the reactions. This experimental system allows for the separation of the individual reaction steps during substrate hydrolysis and the determination of their rate constants dependably. We suggest a refined model for the reaction mechanism, in which acylation is preceded by the reversible formation of the first tetrahedral intermediate. Furthermore, the thermodynamics of these steps were also investigated. The formation of the first tetrahedral intermediate is highly exothermic and accompanied by a large entropy decrease for the wild type enzyme, whereas the signs of the enthalpy and entropy changes are opposite and smaller for the R193G mutant. This difference in the energetic profiles indicates much more extended structural and/or dynamic rearrangements in the equilibrium step of the first tetrahedral intermediate formation in wild type human trypsin 4 than in the R193G mutant enzyme, which may contribute to the biological function of this protease.Trypsin is a prototype of the S1 serine protease family, the largest group of proteases. The residues indispensable for its enzymatic activity, His-57, , are structurally conserved and are referred to collectively as the catalytic triad (1). These residues are located at the active site of the enzyme. As the first step of the catalytic cycle, the enzyme binds the substrate forming the non-covalent Michaelis complex. The hydroxyl oxygen of the catalytic serine makes a nucleophilic attack on the electron-deficient carbonyl carbon of the scissile bond, and a covalent bond is formed. The attacked carbon atom becomes tetrahedrally coordinated; thus this state is called the first tetrahedral intermediate. In the next step, this species breaks down to yield the C-terminal (first) product and the acyl-enzyme. The acylation is followed by deacylation, a mechanistically similar action, in which the acyl-ester bond is attacked by a water molecule that has previously been activated by 3).During the hydrolysis of the scissile bond, tetrahedral intermediates are developed that are stabilized via hydrogen bonding interactions, in which the amido groups of residues Gly-193 and Ser-195 act as H-donors and the developing oxyanion intermediate is the acceptor (4, 5). Despite glycine being the far most convenient amino acid at position 193 for optimal...