Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Enzymes
Serine proteinases

Serine proteinases, named for the characteristic amino acid residue in their active sites, are widespread in nature and, together with Proteolytic Enzymes of other classes (aspartic, Cysteine, and metalloproteinases), ensure the deep Cleavage of Proteins—The basis of their Catabolism—and a range of Limited proteolysis reactions of regulatory significance. Several families unite evolutionarily related serine proteinases. The animal kingdom is particularly characterized by diverse serine proteinases belonging to the Chymotrypsin family, meaning they are structurally and mechanistically similar to chymotrypsin (the most studied enzyme of this type) in the Spatial Structure, architecture, and catalytic center mechanism. Certain streptomycete serine proteinases also belong to this family. Serine proteinases of another family, the subtilisins, are especially typical of prokaryotes, but are also found in plants and animals. A third family is also known: serine Carboxypeptidases produced by animals, Fungi, Yeasts, and plants. The structural uniformity of the catalytic centers and the profound mechanistic similarities among enzymes of these families, which are completely dissimilar in their spatial structures, are apparently explained by the convergent evolution of precursor proteins.

The following Discussion summarizes data on the MECHANISM OF ACTION of serine proteinases.

10.5.1. Substrate Binding

The substrate-binding region in proteinases, which participates in complex formation with proteins or their fragments, is quite extensive. Typically, 5–8 amino acid residues can interact with it—2 to 4 on either side of the cleaved peptide bond. Observing the binding of true substrates—Peptides of considerable length—using X-ray crystallography is not feasible due to the extremely short lifetimes of such complexes, in which the substrate is rapidly cleaved. Therefore, complexes of enzymes with substrate analogues are studied instead. For instance, the interaction of aldehyde peptides (compounds in which the C-terminal residue is replaced by an amino aldehyde) with a chymotrypsin-family enzyme, streptomycete proteinase A, has been described in detail:

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Upon interaction with a serine proteinase, the aldehyde group forms a hemiacetal with the serine hydroxyl of the Active Site. This bond serves as a model for the transient enzyme-substrate complex resulting from the interaction between the hydroxyl group of the active-site serine residue and the carbonyl of the cleaved peptide bond:

The resulting highly stable complex makes it possible to characterize the network of interactions between the enzyme and the substrate at the region preceding the attacked peptide bond. The interactions between the enzyme and the C-terminal part of the substrate have been studied using other models, particularly complexes of enzymes and natural proteinase inhibitors.

In the region preceding the attacked peptide bond, the enzyme forms an antiparallel $\beta$-structure-like arrangement with the substrate, establishing three Hydrogen Bonds. These are supplemented by four additional hydrogen bonds with the Functional groups of the catalytic center. The accommodation of the peptide substrate in the binding site is accompanied by a slight Displacement of the surrounding enzyme regions, as well as the release of a chain of Water molecules that hydrated the functional groups of the free enzyme through hydrogen bonding.

The network of hydrogen bonds between the substrate and the enzyme is complemented by A number of hydrophobic contacts. The most important of these are formed by the side chain of The amino acid residue whose carbonyl group belongs to the peptide bond attacked by the enzyme. According to established nomenclature, this amino acid residue is designated P₁, the preceding one P₂, followed by P₃, P₄, and so on toward the amino terminus of the substrate. The residue whose amino group forms the attacked bond is designated P'₁, the next one P'₂, and so forth. The corresponding Regions of the substrate-binding site are designated S₄, S₃, S₂, S₁, S'₁, S'₂.

Streptomycete proteinase A, which hydrolyzes peptide bonds involving the carboxyl group of hydrophobic Amino Acids—thus having a Specificity similar to chymotrypsin—forms about 40 non-covalent contacts with the side chain of the P₁ residue. The binding of this hydrophobic group is accompanied by the expulsion of water molecules from the hydrophobic S₁ pocket. Such an extensive system of non-covalent contacts assigns particular importance to the P₁ residue, whose nature largely determines the choice of the peptide bond cleaved by serine proteinases—in other words, the specificity of these enzymes, although the latter also depends (albeit to a lesser extent) on neighboring amino acid residues.

Interestingly, residues such as Alanine, Glycine, Threonine, glutamine, and Proline, which line the enzyme's S₁ pocket, participate in forming hydrophobic contacts with the substrate. At first glance, the participation of these mostly hydrophilic amino acids in building a hydrophobic binding site seems paradoxical; however, this reflects the fundamental difference in The behavior of identical residues when free in solution versus when incorporated into a fixed protein spatial structure. In the latter case, even the methylene group of glycine, being separated or isolated from hydrophilic peptide bonds, is capable of behaving as a hydrophobic element. Such small individual elements, held spatially in place by the hydrogen bonds of neighboring groups, form a kind of hydrophobic mosaic that lines the cavity or "pocket" within the substrate-binding site.

Apparently, this method of forming hydrophobic zones is even more efficient than using distinctly hydrophobic amino acids with large side groups, since anchoring the latter within the protein surface structure—specifically restricting their rotational freedom—is more difficult than fixing a small hydrophobic element.

In serine proteinases that specifically cleave peptide bonds formed by the carboxyl groups of Lysine or Arginine—namely Trypsin and its analogues—multiple contacts are also established between the polypeptide chain of the substrate and the enzyme. In this case as well, The formation of a network of hydrogen bonds between the substrate peptide chain and the enzyme is critically important:

The binding of the P₁ residue is largely determined by Electrostatic Interactions between the cationic groups in the substrate side chain—the —NH⁺₃ group in the case of lysine, or the guanidinium group

in the case of arginine—and the carboxylate anion of an aspartic acid residue located at the bottom of the hydrophobic S₁ pocket (Asp-189 in trypsin) (Fig. 10.3). Such electrostatic interaction is favored by the Isolation of the ion pair from contact with water.

Fig. 10.3. Accommodation of the substrate P₁ side chain in the S₁ pocket of trypsin.

A — interactions of the lysine —NH⁺₃ group; B — interactions of the arginine guanidino group. Open circles indicate oxygen atoms, dark circles indicate nitrogen atoms, W represents water molecules held by the enzyme, dashed lines show hydrogen bonds, and numbers correspond to their lengths in angstroms.

Furthermore, this is complemented by a system of hydrogen bonds between the guanidino or amino group of the substrate and the carboxylate ion of Asp-189, as well as oxygen atoms of several C=O groups of the enzyme. Protein Engineering substitution of the Asp-189 residue with a lysine residue in trypsin radically altered the enzyme's specificity: it lost The ability to hydrolyze arginine and lysine peptide bonds, but acquired (albeit weak) activity toward bonds formed by hydrophobic residues such as phenylalanine, leucine, and Tyrosine, thus becoming functionally similar to chymotrypsin, another enzyme of the same family.

Sometimes the P₁ residue is less dominant in determining specificity, and interactions with other amino acid residues of the substrate become more significant. For example, the specificity of kallikrein—a trypsin analogue involved in animal regulatory mechanisms—is determined by the presence of arginine at the P₁ position and a hydrophobic residue at the P₂ position of the substrate, thus making its specificity much narrower.

When proteinases act on native protein substrates, the specificity of Hydrolysis is determined not only—and often not even primarily—by the Amino Acid Sequence of the cleaved region, but rather by its spatial, steric accessibility. For instance, the embedding of kallikrein's entire active site within a cleft, brought about by the elongation of adjacent peptide loops of the enzyme, makes the hydrolysis of conventional protein substrates practically impossible, even if they contain the hydrophobic residue–arginine sequence. Kallikrein attacks only those proteins in which the sequence matching its specificity is positioned on a protruding region of the substrate protein that is complementary to the cleft of the enzyme's active site. This achieves the exceptionally narrow, almost unique specificity of kallikrein—an enzyme involved in releasing vasoactive peptides from protein precursors while remaining virtually inactive toward other proteins.

Let us note one more feature of protein substrate binding in the active center of proteinases. During the formation of complexes with sufficiently long peptides spanning the active site, the conformation of the polypeptide chain within the binding region is believed to undergo a certain degree of "twisting," slightly distorting the planar STRUCTURE OF THE peptide bond targeted by the enzyme. This creates favorable conditions for its transition from a planar to a pyramidal configuration close to the Transition State. Consequently, even at the Michaelis complex formation stage during substrate binding, a stereochemical transition of key importance for efficient catalysis is already primed.

Substrate-binding features characteristic of serine proteinases—such as multiple interactions, precise orientation relative to the catalytic center groups, and the preferential binding of a conformation stereochemically close to that of the transition state—are typical of many enzymes. It must be emphasized that the two crucial stages of catalysis, substrate binding and the catalytic conversion itself, although considered separately, are in reality merged into a single unified mechanism.

10.5.2. Catalytic Mechanism

The Topography of the catalytic center in serine proteinases has been described in considerable detail. Studies of The structure of enzyme complexes belonging to this class with various substrate analogs and inhibitors, combined with data on catalytic kinetics, chemical modification of individual enzyme groups, NMR spectroscopy, and protein engineering, have led to the following concepts regarding The Mechanism of action of serine proteinases.

The primary role in substrate transformation is played by the hydroxyl group of Ser-195 (here and hereafter, residue numbering follows The sequence of the most thoroughly studied enzyme, $\alpha$-chymotrypsin), the imidazole ring of His-57, the $\beta$-carboxyl group of Asp-102, and the peptide groups of the Ser-195 and Gly-193 residues. Thus, in serine proteinases, as in Other Enzymes, the active center is assembled from amino acid residues located in completely different regions of the polypeptide chain but brought into close proximity within the three-dimensional structure of the enzyme (Fig. 10.4).

Fig. 10.4. Spatial arrangement of functional groups in the catalytic center of serine proteinases.

The Components of the catalytic center are shown—namely, the serine, Histidine, and aspartic acid residues; nitrogen and oxygen atoms participating in the catalytic mechanism are shown in black. Dashed lines indicate hydrogen bonds. Numbering is based on the amino acid sequence of chymotrypsin; in other serine proteinases, these amino acids may occupy different positions in the Primary Structure, but their relative spatial arrangement is strictly conserved.

As a rule, the components of the catalytic center interact with neighboring enzyme groups, which locks them into a specific orientation and alters their chemical characteristics. For instance, the carboxyl group of the Asp-102 residue forms four hydrogen bonds with neighboring groups, stabilizing its anionic form and consequently shifting the $\text{p}K_a$ of this residue to 2–3 instead of 3.6 (the value typical of a standard aspartic acid $\beta$-carboxyl group). The proximity of this carboxylate ion stabilizes a proton on one of the nitrogen atoms of the histidine imidazole group—specifically the one facing Asp-102—while simultaneously defining the specific electronic structure of the His-57 imidazole ring. The latter feature is highly characteristic of functional groups in proteins and has no parallel in the behavior of similar groups, particularly imidazole, in small molecules. The local environment of functional groups in a protein is anisotropic.

Although the Influence of the microenvironment on the behavior of functional groups within an enzyme is substantial, the reactivity of the active center components in serine proteinases in the absence of a substrate does not exhibit drastic differences that exceed the normal variations in Chemical properties of functional groups on a protein surface. Thus, reports suggesting that the His-57 imidazole and Asp-102 carboxyl in the protein's active center had seemingly swapped properties, with the imidazole turning out to be more acidic than the carboxyl group, proved to be erroneous. Likewise, one should not assume that a Relay transfer of a negative charge occurs in the free enzyme from the Asp-102 carboxylate ion to Ser-195, converting the latter's hydroxyl into a hydroxide ion—an extremely strong nucleophile. Such a charge transfer would be instantly nullified by the reaction of the hydroxide ion with water:

In contrast, within the enzyme-substrate complex, The chemical properties of the participants in the catalytic process—including the functional groups of the catalytic center—change dramatically. This is driven not only by the extensive network of non-covalent interactions encompassing the enzyme-substrate complex, but also by the complete insulation of the system from water. As M. Dewar aptly noted, the very process of enzyme-substrate binding requires, as a necessary prerequisite, the displacement of water molecules that surround the free substrate and fill the active center region of the enzyme. Consequently, the catalytic reaction is transferred to an entirely different environment. This consideration once again underscores the inseparable link between the two facets of the biocatalytic process: substrate binding and its actual transformation.

A hypothetical mechanism of action for serine proteinases can be described as follows (Fig. 10.5). It should be borne in mind that sequentially described processes may in fact occur synchronously.

The binding of the polypeptide substrate within the active center positions the carbonyl group of the targeted peptide bond such that its oxygen atom resides in the so-called oxyanion hole. The formation of two hydrogen bonds between this oxygen atom and appropriately oriented $\text{N-H}$ groups belonging to the peptide bonds of Ser-195 and Gly-193 promotes the polarization of the $\text{C=O}$ bond, shifting it toward $\text{C-O}^-$ and stabilizing the oxyanion. Due to precise substrate binding by the enzyme, the positive charge on the carbonyl carbon is brought into close spatial proximity with the oxygen atom of the serine hydroxyl group. This induces polarization of the hydroxyl group, adopting a structure close to $-\text{CH}_2-\text{O}^- \dots \text{H}^+$, which sharply enhances the nucleophilicity of the serine oxygen atom.

Fig. 10.5. Mechanism of action of serine proteinases.

A — functional groups in the active center of the enzyme. The left hemisphere shows the histidine imidazole backed by the aspartate carboxyl, anchored by a system of hydrogen bonds; the right hemisphere shows the serine side chain and the two hydrogen bonds of the oxyanion hole.

B — in the right hemisphere, the product of substrate interaction (tyrosine at the $\text{P}_1$ position is shown); the oxygen atom is positioned in the oxyanion hole; the proton that departed from the serine hydroxyl group moves toward the imidazole nitrogen and subsequently toward the nitrogen atom of the cleaved

peptide bond.

C — a water molecule has positioned itself near the carbonyl carbon atom, with its proton transferred to the nitrogen of the histidine imidazole.

D — the hydroxyl group of water has added to the carbonyl carbon atom, completing the Cleavage of the peptide group.

Thus, in the scheme under consideration, the enhancement of serine nucleophilicity occurs under METABOLISM/18.html">The Influence of the substrate carbonyl, which is properly oriented and polarized via interaction with the enzyme; the substrate also shields the nucleophile, protecting it from water.

Next, a covalent bond forms between the substrate carbon and the substrate oxygen, which is likely longer and less stable than usual (Fig. 10.5, B):

The proton cleaved from the serine hydroxyl travels along a trajectory dictated by the His-57 imidazole group, approaching the unprotonated nitrogen atom, and is subsequently transferred to the $\text{-NH-}$ group of the scissile peptide bond. As is well known, the nitrogen atom in peptide groups is virtually devoid of basic properties due to the displacement of the lone electron pair involved in the $\text{C-N}$ bond, which possesses partial double-bond character. However, following the polarization of the carbonyl group within the oxyanion hole, the planar geometry of the peptide bond is lost, the $\text{C-N}$ bond becomes a single bond, and the nitrogen atom acquires The properties of a very strong base. Consequently, a proton from the imidazole group—a weak base—is transferred along a curved trajectory to the $\text{-NH}^-$ group. The $\text{C-N}$ bond breaks, and the peptide substrate fragment is released (Fig. 10.5, C).

Thus, the first task of catalysis is accomplished: the peptide bond is cleaved, and the C-terminal fragment of the peptide is released. The remaining part of the peptide (from the P₁ residue to the amino terminus) temporarily remains linked to the enzyme via the serine residue. Apparently, under certain conditions, this bond can be stabilized through the reduction of the carbonyl group and the mutual compensation of positive and negative charges, such that the peptide fragment forms an ester bond with the enzyme, resulting in the Formation of the so-called acyl-enzyme:

When serine proteinases act on certain substrate analogues, it is possible to observe and sometimes even isolate a sufficiently stable acyl-enzyme. However, during the hydrolysis of conventional substrates, such stabilization is apparently not strictly necessary, and it is the derivative with the polarized carbonyl group that undergoes hydrolysis. In any event, following the departure of the C-terminal peptide fragment, a water molecule occupies the vacated space and positions itself to form a Hydrogen bond with the nitrogen of the imidazole group, while its oxygen atom comes into close proximity to the positively charged carbon atom of the carbonyl group. This leads to the abstraction of a proton—which attaches to the imidazole—and the formation of a hydroxyl ion that readily interacts with the positively charged carbonyl carbon. The proton migrates to the serine oxygen, resulting in the cleavage of the C—O bond, the restoration of the active-site hydroxyl group, and the release of the N-terminal peptide fragment, thereby completing the catalytic cycle (Fig. 10.5, D):

This stage of the reaction proceeds overall in a manner analogous to the first, except that a water molecule assumes The Role of the serine hydroxyl group.

Assuming that the intermediate product is stabilized and an acyl-enzyme is formed, the mechanism of its hydrolysis will be practically identical. One need only assume the polarization of the C = O bond in the ester group of the acyl-enzyme via hydrogen bonds within the oxyanion hole, which leads to the generation of a positive charge on the carbon atom of the carbonyl group—that is, to the formation of the exact same intermediate whose fate was traced above.

A crucial feature of the examined mechanism of serine proteinase action, which appears typical for other enzymes as well, is the stabilization of the tetrahedral transition state within the enzyme-substrate complex. It is characteristic that alkylboronic acids serve as highly potent inhibitors of serine proteinases; these compounds possess a pre-existing tetrahedral configuration of substituents—three hydroxyl groups and an alkyl residue—that mimics the structure of the transition state. Particularly effective are derivatives of the boronate analogue of phenylalanine, which binds to the S₁ subsite via its benzyl group and interacts with the catalytic center; the corresponding inhibition constant K_i is 10^-8 M:

Aldehyde peptides, as mentioned earlier, act as inhibitors of serine proteinases by forming a covalent hemiacetal bond with the active-site serine—a structure that is, to a certain extent, analogous to the acyl-enzyme. In this process, the free hydroxyl group binds within the oxyanion hole in the exact same manner as the carbonyl oxygen does in the enzyme-substrate complex.

Serine proteinase inhibitors that undergo The first phase of the catalytic process upon interacting with the enzyme to form an acyl-enzyme are well documented. While some such derivatives undergo hydrolysis at a measurable rate, others—specifically arylsulfonyl fluoride derivatives and a number of organophosphorus compounds—prove to be stable, leading to the irreversible inhibition of the enzyme. The structures of the stable acyl-enzymes resulting from the inhibition of serine proteinases by phenylmethylsulfonyl fluoride (left) and diisopropyl fluorophosphate (right) are shown below:

The concepts regarding the mechanism of action of serine proteinases outlined above have been confirmed using protein engineering Methods. Substituting the active-site serine with alanine in a number of enzymes within this family (via Site-Directed Mutagenesis) invariably leads to an almost complete loss of activity. Replacing components of the catalytic center in subtilisin (a bacterial proteinase) yielded the following results: replacing Ser-221 (the residue corresponding to Ser-195 in chymotrypsin) with alanine reduced the enzyme's activity by a factor of approximately 10⁶, with virtually the entire effect attributable to a drop in the catalytic constant k_cat, whereas the Michaelis constant remained unchanged. Similar results were obtained when the catalytic histidine was replaced with alanine. The decrease in k_cat upon replacing Asp-32 with alanine was somewhat smaller, yet still very substantial, with k_cat in this case amounting to 4∙10^-6 of the value characteristic of the wild-type enzyme.

Thus, all components of the catalytic center "triad" are critically important for efficient catalysis. The role of the aspartic acid residue, which might seem less significant in light of the mechanism described above, apparently lies in the fact that the carboxylate anion of this residue, first, spatially fixes the imidazole ring of the catalytic histidine and, second, ensures the stabilization of the tautomeric state of the imidazole group depicted in the schemes above.

Analyzing the role of the N—H groups that form the oxyanion hole using this method is more challenging, since in chymotrypsin and its homologous enzymes both groups belong to the peptide backbone. However, in subtilisin, the amide group of asparagine performs the function of one of the components of the oxyanion hole. Replacing this residue with leucine diminishes the polarizing efficiency of the oxyanion hole toward the substrate's C = O group, resulting in an activity drop of approximately 10³-fold, while nevertheless retaining a quite significant level of activity.