BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 8. ZYMOGEN ACTIVATION: DIGESTIVE ENZYMES AND BLOOD CLOTTING FACTORS

8.11. Trypsin and Elastase: Variations on a Theme

Trypsin and Elastase are similar to Chymotrypsin in many respects.

1. All three Enzymes are secreted by the Pancreas as zymogens and are activated by the Cleavage of a single peptide bond. The resulting terminal amino group folds into the interior of the molecule and forms an electrostatic bond with the carboxylate ion of aspartate-194.

2. The Amino acid sequences of these three enzymes are about 40% identical. For amino acid residues located in the interior of the molecule, the degree of identity is even higher.

3. Fluorophosphates, particularly DIPF, inhibit all three enzymes. Like chymotrypsin, both trypsin and elastase contain a Serine residue in their Active Site. Furthermore, The sequence of Amino Acids surrounding this serine is identical in all three enzymes: Gly-Asp-Ser-Gly-Gly-Pro.

4. X-ray crystallographic analysis revealed a high degree of similarity in the tertiary structures of these three enzymes (Fig. 8.21). Like chymotrypsin, both elastase and trypsin possess a charge-Relay system and an oxyanion hole.

Class="center">Fig. 8.21. Comparison of the polypeptide backbone Conformations of chymotrypsin (A) and elastase (B). The locations of the charge-relay system (residues 102, 57, and 195) and the α-amino group of residue 16 are highlighted in color to emphasize the structural similarity of these enzymes

5. The Catalytic Mechanism of all three enzymes is nearly identical. Their efficiency as catalysts is due to their stabilization of the Transition State of the reaction. The oxyanion hole and the charge-relay system are the essential Structure/83.html">Structural elements of each enzyme that facilitate The formation of the tetrahedral intermediate.

While similar in structure and MECHANISM OF ACTION, these enzymes differ strikingly in their Specificity. The substrate for chymotrypsin must have an aromatic or a large nonpolar side chain. Trypsin requires a substrate containing Lysine or Arginine. Neither of these substrates is suitable for elastase, which shows specificity for small, uncharged side chains. As shown by X-ray crystallographic analysis, this difference in specificity is due to very minor structural differences in the substrate-binding sites (Fig. 8.22). In chymotrypsin, aromatic

and large nonpolar side chains of the substrate are accommodated in a nonpolar pocket. Trypsin has a similar pocket, which differs, however, from the nonpolar pocket of chymotrypsin by a single residue substitution: an aspartate is present instead of serine. This aspartate in the trypsin pocket forms a strong electrostatic bond with the positively charged lysine or arginine side chains of the substrate. In elastase, there is no such substrate pocket because the two Glycine residues lining the pocket in chymotrypsin are replaced in elastase by much larger valine and Threonine residues.

Fig. 8.22. A highly simplified representation of the substrate-binding site in chymotrypsin, trypsin, and elastase

The structural changes that occur during trypsinogen activation differ somewhat from those associated with chymotrypsinogen activation. X-ray crystallographic studies, carried out independently by Robert Huber and Robert Stroud, showed that trypsinogen activation involves significant Conformational Changes in four extended segments of the polypeptide, comprising about 15% of the molecule. These regions, termed activation domains, lack a stable structure in the zymogen but acquire a highly defined conformation in trypsin. Furthermore, the oxyanion hole in trypsinogen is located too far from Histidine-57 to facilitate the Formation of the tetrahedral intermediate.

8.12. Pancreatic Trypsin Inhibitor Binds Tightly to the Active Site of Trypsin

The regulation of pancreatic protease activity occurs in two distinct ways. The first is The conversion of a zymogen into an active protease by the cleavage of a single peptide bond. This is a highly precise mechanism for "switching on" enzymatic activity; however, it is irreversible, and therefore a second regulatory mechanism must exist to halt proteolysis. This function is performed by specific protease inhibitors. For example, pancreatic trypsin inhibitor, a 6-kDa protein, inhibits trypsin activity by binding extremely tightly to its active site (Fig. 8.23). The dissociation constant of the complex is 10^-13 M, which corresponds to a Standard Free energy of binding of approximately -18 kcal/mol. A remarkable feature of this interaction is that the complex does not dissociate in either 8 M urea or 6 M guanidine hydrochloride. Yet, these Denaturing Agents almost always cause the dissociation of protein oligomers into their constituent subunits. This astonishing Stability of the complex is explained by the fact that the trypsin inhibitor is a highly effective substrate analog. X-ray crystallographic analysis showed that the side chain of lysine-15 in the inhibitor molecule binds to the side chain of aspartate in the substrate-specificity pocket of the enzyme. In addition, numerous Hydrogen Bonds are formed between The polypeptide chains of the enzyme and the inhibitor. They are arranged in the same way as hydrogen bonds between the enzyme and a true substrate. Most importantly, the carbonyl group of lysine-15 and its surrounding atoms in the inhibitor fit tightly into the oxyanion hole of the enzyme. However, the entire complex seems to freeze at this intermediate stage of catalysis—apparently because histidine-57 of the enzyme, being immobile, is unable to transfer a proton to the amide nitrogen of the inhibitor. The peptide bond between lysine-15 and Alanine-16 in the pancreatic trypsin inhibitor is cleaved, but at an extremely slow rate. The half-life of the trypsin-inhibitor complex is several months. The inhibitor has a very high affinity for trypsin due to the near-perfect complementarity of its structure to the Active Site of trypsin. The limited conformational flexibility of the inhibitor at its binding site blocks the catalytic process and leads to the exceptionally high stability of the trypsin complex.

Fig. 8.23. The key feature of the interaction between pancreatic trypsin inhibitor and trypsin is the formation of an electrostatic bond between lysine-15 of the inhibitor and aspartate-189 of the enzyme. In addition, the —NH₃⁺ group of lysine-15 is hydrogen-bonded to several oxygen atoms in the substrate-specificity pocket of the trypsin molecule