Principles of Protein Structure - H. Schultze 1982

Covalent Structure of Proteins
Enzyme-Controlled Modifications of the Main Chain
Activation of Trypsinogen

Trypsinogen is converted into Trypsin, which in turn activates many Other Enzymes. Specific proteases have not been detected in many systems. A classic example of a thoroughly studied Limited proteolysis process is the activation in the digestive tract of zymogens [1, 138, 139], which are inactive enzyme precursors [143]. The protease trypsin is considered a key digestive enzyme [1], not only because of its intrinsic activity toward dietary Proteins, but also because it acts as the sole activator of other zymogens, notably chymotrypsinogens, procarboxypeptidase, proelastase, and phospholipase A2 (prophospholipase A). Trypsin itself is secreted into the duodenum as its inactive precursor, trypsinogen (Fig. 4.5).

The trypsinogen activator recognizes a specific sequence. The physiological activator of trypsinogen, enteropeptidase (also known as enterokinase), is located on the membrane surface of epithelial Cells in the duodenum. The remarkably narrow Specificity of enteropeptidase is highlighted by the fact that this enzyme does not hydrolyze a single bond in any native or denatured protein tested, with the sole exception of the specific bond in trypsinogen (or in synthetic Peptides) located immediately upstream of the Asp-Asp-Asp-Asp-Lys sequence (Neurath, private communication, cited in [138]). Thus, the specificity of this enzyme is determined by the Amino Acid Sequence rather than the tertiary Structure.

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Fig. 4.5. Amino acid sequence of canine protrypsinogen (preliminary data [153]).

It is hypothesized that Met serves as the N-terminal amino acid for all nascent eukaryotic polypeptide chains (subsequently being cleaved off). It remains unclear which residue of protrypsinogen, 16 or 17, corresponds to the N-terminus of canine trypsinogen. The method used does not allow for a clear distinction between Leu and Ile residues (e.g., at position 24).

Neglecting correction factors for the natural Abundance of individual Amino Acids [144, 145], the Asp-Asp-Asp-Asp-Lys sequence occurs once every 20⁵ combinations. This demonstrates that a unique amino acid sequence provides a much more reliable site for specific protein recognition than any unique feature of tertiary structure—a high degree of reliability being essential for any initiator of an enzymatic cascade.

Trypsinogen activation is characteristic of other systems. The trypsinogen–enteropeptidase system appears to be a typical model of specific proteolysis (Table 4.2) in the following respects:

a) in most cases, the transformation (proprotein → protein) can be achieved by trypsin in vitro, albeit at a relatively low rate and with limited specificity [146]. It should be emphasized that active trypsin occurs in vivo exclusively within the duodenum.

b) The enzyme responsible for the in vivo conversion is far more efficient and specific; each of the reactions listed in Table 4.2 apparently requires a distinct, dedicated enzyme. One would expect that, much like enteropeptidase, specific proteases recognize not merely a single key residue, but a broader structural motif (such as the Asp-Asp-Asp-Asp-Lys sequence found in trypsinogen).

c) Specific proteases are frequently membrane-bound. Complement action—the result of the protective "complement" protein system—is part of the body's immune defense mechanism that eliminates foreign cells, such as Bacteria or Cancer cells. The Complement System itself is composed of at least 18 different Plasma Proteins [147]. Functionally, the majority of these proteins are proproteins (zymogens) acting as precursors in a sequential or simultaneous enzymatic cascade that provides signal Amplification. The entire complement system, with all its components at optimal concentrations, is constantly primed for action and can be triggered at a moment's notice. To fulfill its biological role—providing defense and activating numerous protein precursors—the complement pathway is closely intertwined with the Blood Coagulation SYSTEM [148].

Fig. 4.6. Diverse Functions of complement protein C3 and its Cleavage products [147].

The cleavage site for C3 convertase on C3 is marked with an upward-pointing arrow. C3 ($M_r$ 180,000) participates in the alternative (properdin) pathway and also serves as the physiological precursor for two active fragments: the smaller C3a and the larger C3b. C3a is involved in inflammatory and signaling responses, whereas C3b acts as a subunit or modulator for four distinct complement enzymes. Notably, the inactivation of both C3a and C3b is also governed by limited proteolysis.

Proteolytic triggering offers clear physiological advantages. At THE MOLECULAR LEVEL, complement proteins exhibit many hallmark features of specific proteolytic cascades [138].

a) Much like individual proteolytic reactions that proceed quantitatively and irreversibly, the entire complement cascade can only flow in a single direction.

b) The activation and inactivation of proteins—entailing rapid conformational shifts at active binding sites—impose strict temporal and spatial constraints; consequently, complement activity is localized strictly to the immediate microenvironment of IMMUNOGLOBULINS bound to target cells.

c) Both the parent protein and its cleavage products can exert distinct physiological functions; specific proteolysis orchestrates and regulates all of these activities. This is clearly exemplified by complement factor C3 (Fig. 4.6).

d) Complement activation also has a morphogenetic dimension. A sequence of specific proteolytic events drives molecular aggregation, resulting in the stable assembly of five distinct soluble proteins onto The Cell membrane [147].