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

Post-translational protein modification
Limited proteolysis

Limited proteolysis is a crucial reaction in the Post-translational Modification of Proteins. Ordinary “unlimited” or deep proteolysis&mdashthe Cleavage of a protein by proteinases&mdashproceeds by an “all-or-none” principle. As a rule, a compact protein is relatively resistant to the action of proteinases because it virtually lacks the extended “open” segments of the polypeptide chain required for binding Proteolytic Enzymes. Therefore, the initial attack is limited to the cleavage of individual loops and terminal segments of the protein. However, if an “internal” segment of the polypeptide chain undergoes proteolysis after becoming accessible due, for instance, to the partial (and reversible) Denaturation of the protein globule, the Spatial Structure becomes destabilized, the substrate protein undergoes irreversible denaturation, and the entire polypeptide chain becomes vulnerable to proteolytic attack. This results in deep Hydrolysis yielding short fragments. Under such a pathway of proteolysis, the hydrolysate contains the initial protein (whose amount steadily decreases) and low-molecular-weight peptide products (whose content continuously increases), whereas intermediate products are generally not observed.

Limited proteolysis, in which the process is delayed or stops completely at intermediate products, is possible only when the proteolysis products retain their compactness—for example, upon the cleavage of a fragment from either end of the peptide chain, the rupture of a loop on the globule surface, or the Cleavage of the peptide chain between sufficiently stable Protein domains.

The course of limited proteolysis and its outcome depend crucially on the Structural Features of the substrate protein. Consequently, identical or very similar results are often achieved using different proteinases whose sites of action are determined by the spatial architecture of the substrate. If The sequence of amino acid residues in the cleavage-permissive region (the hinge region) matches the Specificity of the attacking proteinase, the efficiency of the process increases. This is precisely how Post-translational protein modification by proteinases operates.

Let us examine several typical processes of limited proteolysis.

11.8.1. Formation of a Non-Canonical Cell/13.html">Protein Structure: Insulin Biosynthesis

Insulin, composed of two polypeptide chains, A and B, containing 21 and 30 amino acid residues respectively and linked by Disulfide Bonds, is too small to form a stable globular structure with sufficient efficiency. Indeed, insulin can be obtained by the recombination of separately synthesized A and B chains, where the Cysteine residues undergo oxidation to form The system of disulfide bonds characteristic of the native protein. However, this process is slow and typically yields a relatively low amount of insulin.

The tertiary structure is formed much more easily by the insulin precursor, proinsulin, in whose polypeptide chain the “future” A and B chains of insulin are connected by peptide C in the sequence B–C–A. Such a protein, containing about 90 amino acid residues in a single polypeptide chain, folds into a compact structure, followed by the closure of disulfide bridges that stabilize the molecule as a whole; subsequently, a specialized proteinase “excises” peptide C. The cleavage sites of this proteinase are predetermined by two factors: the Spatial structure of proinsulin and the presence of two signals in its polypeptide chain—namely, two pairs of cationic Amino Acids arranged in the sequence as follows:

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The proteinase Processing such a precursor recognizes pairs of amino acids with cationic side chains, such as Arg–Arg, Lys–Arg, etc., and cleaves the peptide bond at the C-terminus of these pairs. As a result, peptide C is excised along with the Lys–Arg sequence at its C-terminus, liberating the A and B chains, with the C-terminus of the B chain retaining two Arginine residues that must be removed. This is carried out by a specialized metallocarboxypeptidase with an optimal activity in a mildly acidic environment.

It should be noted that the combined action of a proteinase that hydrolyzes peptide bonds following a pair of cationic amino acids (such enzymes have been found in animals and Yeast) and a carboxypeptidase that subsequently removes these amino acids (similar in specificity to carboxypeptidase B) is utilized in The biosynthesis of short Peptides, such as enkephalin. The specificity of this system is quite high, and the processing of long precursors apparently occurs even before the stable spatial structure is formed. For instance, the polypeptide chain of proopiomelanocortin, comprising 235 amino acid residues, is cleaved by this pathway at seven sites to yield endorphin, adrenocorticotropic hormone, α- and γ-melanocyte-stimulating Hormones, and other physiologically active peptides.

11.8.2. Disjunction of Protein Globules in Polyproteins

In A number of cases, several structurally and functionally distinct proteins are initially synthesized as a single very long polypeptide chain, whose individual segments fold independently into protein globules to form what is known as a polyprotein. This is how FATTY ACID BIOSYNTHESIS enzymes are synthesized, for example. The synthesis of polyproteins is particularly characteristic of Retroviruses. Subsequently, specific proteinases cleave the peptide links between individual globules, after which the latter function independently. In particular, the Components of the HUMAN IMMUNODEFICIENCY VIRUS polyprotein are separated in this manner. Apparently, the precision of peptide link cleavage in the polyprotein is so critical in this case that the process cannot always be entrusted to host cell proteinases. The polyprotein contains a highly specific human immunodeficiency virus proteinase, distantly related to Pepsin, which recognizes characteristic Amino acid sequences within the links and cleaves them precisely. It is hypothesized that the selective inhibition of such proteinases could serve as a strategy to combat viral infection.

11.8.3. Domain Separation

This reaction is utilized, in particular, as a necessary stage in the MECHANISM OF ACTION of a number of protein toxins produced microorganisms. For instance, diphtheria toxin is synthesized as a protoxin composed of a single polypeptide chain that forms at least two structurally and functionally autonomous domains. One of these is responsible for binding the toxin to the membrane receptor of the target cell, while the other Functions as an enzyme catalyzing The transfer of the adenosine diphosphate ribose moiety of NAD to elongation factor EF, which is thereby inactivated. The toxin–receptor complex, formed by the binding of the first domain to the receptor, is engulfed into a membrane-bounded vesicle via internalization.

To be able to act on the elongation factor EF, the catalytic domain must leave the vesicle and enter the Cytoplasm. Although the toxin possesses a structure that enables the catalytic domain to cross the membrane, it remains tethered to the “recognition” domain even after translocation. Consequently, it cannot leave the vesicle until a proteinase of appropriate specificity cleaves the interdomain region of the polypeptide chain. This releases the catalytic domain and allows it to approach factor EF.

Cleavage of the peptide bond between the α- and β-subunits of the Insulin Receptor is necessary for the activation of this protein. The connecting (hinge) region of the polypeptide chain contains a very characteristic signal sequence, Arg–Lys–Arg–Arg–Xaa, in which the Arg–Xaa bond is cleaved by a specific proteolytic enzyme. Carriers of an abnormal insulin receptor Gene have been identified in which this sequence is replaced by Arg–Lys–Arg–Ser–Xaa. The substitution of Arg by Ser renders the protein resistant to the processing proteinase; the domains fail to separate, resulting in The formation of an inactive receptor. Homozygous carriers of such a mutant gene suffer from diabetes that cannot be compensated by insulin.

Interestingly, an analogously structured signal sequence of four cationic amino acids governs the proteolytic activation of Influenza virus hemagglutinin as well as the maturation of the human immunodeficiency virus surface glycoprotein.

11.8.4. Activation of Enzyme Precursors

Many enzymes, particularly Hydrolases, are synthesized as inactive precursors known as proenzymes or zymogens. The activation of pancreatic Serine proteinase precursors—trypsinogen and chymotrypsinogen—has been studied most extensively. In both cases, the peptide chain of the enzyme is extended at the amino terminus by several amino acid residues, forming an activation peptide whose proteolytic removal converts the virtually inactive zymogen into an active proteinase. For example, the amino-terminal sequence in bovine trypsinogen has the following structure:

The hydrolysis of specifically the Lys–Ile peptide bond by Trypsin is determined primarily by its accessibility: although the trypsinogen molecule contains a number of peptide bonds formed by arginine or Lysine residues that are attacked by trypsin in the denatured zymogen, this particular bond is the first to be cleaved in the native protein. The Activation of Trypsinogen yields trypsin, which is capable of activating other proenzyme molecules, making the overall reaction autocatalytic and characterized by a sharp acceleration.

It is characteristic that The rate of activation provided by trypsin in vivo is nevertheless insufficient, and the activation is initiated by a specialized proteolytic enzyme, enteropeptidase (enterokinase), which is present in duodenal secretion and activates trypsinogen approximately 1000 times faster than trypsin. Such high specificity of enteropeptidase is due to the fact that the enzyme recognizes a cluster of four aspartic acid residues preceding the lysine residue in trypsinogen.

The cleavage of the activation peptide from the trypsinogen molecule triggers a cascade of Changes in the spatial STRUCTURE OF THE activation product. Similar conformational changes are undergone by the activation product of chymotrypsinogen, although the activation peptide in this case looks entirely different and remains covalently linked to the enzyme via a disulfide bond. The amino group of the isoleucine residue (amino-terminal in trypsin) liberated upon activation becomes protonated. The cationic ammonium group is pulled into the interior of the enzyme globule due to the tendency of the hydrophobic side chains of the isoleucine and valine residues to minimize their contact surface with Water.

This process is facilitated by the formation of an ion pair between the α-ammonium group of isoleucine and the negatively charged carboxylate ion of the side chain of the Asp-194 residue, which results in the disruption of the Asp-194–His-40 ion pair that existed in the zymogen. The Displacement of the carboxyl group of the Asp-194 residue leads to local structural rearrangements near the substrate-binding pocket; in particular, the Formation of the hydrophobic pocket is completed through the movement of the side chain of the Met-192 residue. Minor refinements in the positioning of the Functional groups of the catalytic center likely also play a significant role. Note that this center, at least in a rough approximation, pre-exists in the proenzyme, which exhibits (toward small substrates) about 0.01% of the activity characteristic of active trypsin.

The activation of trypsinogen is accompanied, so to speak, by the fixation and more precise Organization of approximately 15% of its spatial structure: the corresponding atoms undergo substantial fluctuations in the proenzyme molecule but become locked in place in trypsin. Thus, the cleavage of the Lys–Ile peptide bond triggers a highly complex mechanism of local rearrangements that activates the molecule. Following the described initial reaction, which dictates the formation of the active enzyme, the hydrolysis of other peptide bonds is observed. This leads to the formation of new molecular forms of trypsin that sometimes differ slightly from the primary activation product in their functional properties, such as the details of their specificity. The activation of chymotrypsinogen proceeds similarly and is also initiated by trypsin. Subsequent cleavage of certain bonds in active Chymotrypsin by other molecules of the enzyme yields a whole set of molecular forms of the enzyme.

A similar mechanism underlies the activation of Blood Coagulation proteinases, where the proteolysis of a single polypeptide chain exposes the α-amino group of the N-terminal residue within the catalytic domain of the active enzyme, presumably triggering a similar chain of structural changes that activates the enzyme.

The difference is that the amino-terminal region of the proenzyme—often a very large structure—remains linked to the catalytic core proper through disulfide and, likely, non-covalent interactions. This imparts novel functional features to the proteinase, such as The ability to bind to Membrane Lipids via Calcium Ions mediated by γ-carboxyglutamic acid residues (see Section 11.3).

In the precursors of other proteinases, such as pepsinogen and procarboxypeptidase, the peptide cleaved off during activation (by pepsin or trypsin, respectively) comprises about 40 amino acid residues and appears to act as a kind of shield covering the active center of the enzyme. The removal of this shield does not require strictly specific proteolysis, unlike the Serine proteinases considered above, and can result from the cleavage of any peptide bond in the sequence connecting the activation peptide to the future enzyme. This leads to non-specific activation, which may yield a mixture of active enzymes differing in length and amino-terminal residues (the so-called frayed ends).

The Biological Significance of proenzyme activation is clear: it allows for the accumulation of significant amounts of a precursor that can be converted into an active enzyme extremely rapidly through a single reaction—the cleavage of a single peptide bond. De novo synthesis of substantial amounts of the enzyme would require considerably more time, which is disadvantageous, especially in digestive processes. At the same time, accumulating active proteinases inside The Cell could prove highly detrimental. Biosynthesis as a proenzyme helps circumvent this problem as well.

It should not be assumed that zymogen activation is exclusive to animal enzymes; virtually all bacterial proteinases are known to be synthesized as precursors.