Chemistry and Biology of Proteins - F. Haurowitz 1953

Proteins with Enzymatic Properties
Enzyme Specificity

When examining The Nature of Enzymes and their components, one must always bear in mind that the presence of enzymes is detected solely through their action on a corresponding substrate. To determine Enzyme Specificity, it is necessary to investigate its action on various substrates that differ from one another only in certain FEATURES OF MOLECULAR Structure. This research methodology was notably advanced by Bergmann and his coworkers, whose work was of exceptional importance in clarifying The Mechanism of proteolytic action. Prior to their studies, it remained unknown precisely which peptide bonds are cleaved by Pepsin, Trypsin, and other Proteolytic Enzymes. Using a method they developed, Bergmann and his collaborators [21] synthesized A large number of various Peptides and employed them as substrates for proteolytic enzymes. As a result of these investigations, it was established that trypsin predominantly cleaves peptides containing the basic Amino Acids Arginine or Lysine, whereas pepsin acts mainly on peptides containing the aromatic amino acid Tyrosine [22]. These findings led to the Conclusion that the alkaline side chains of arginine or lysine specifically interact with molecular groups located On the surface of trypsin, whereas The structure of the aromatic ring of tyrosine corresponds to the surface architecture of pepsin.

The fact that enzymes hydrolyze only natural l-peptides indicates that the stereochemical configuration of the substrate is critical for enzyme-substrate complex formation. To account for the stereochemical specificity of enzymes, one must assume that the binding between the enzyme and the substrate involves three or more distinct atomic or molecular groupings [23]. Obviously, such binding can occur only when The surface of the enzyme comes into exceptionally close contact with the surface of the substrate, which presumably requires a precise geometric fit between both surfaces.

It was formerly assumed that proteolytic enzymes were capable of hydrolyzing only —CONH— peptide bonds. Recently, however, Neurath, Schwert, and their coworkers demonstrated that proteolytic enzymes can also hydrolyze —СО—О— ester bonds.

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Thus, for instance, trypsin cleaves benzoyl-L-arginine methyl ester more rapidly than benzoyl-L-arginine amide [24], while carboxypeptidase hydrolyzes hippurylphenyllactic acid and the peptide hippurylphenylalanine at identical rates [25].

These experiments severely challenged the foundations of Enzyme Classification. Since enzymes can only be classified based on their action upon specific substrates, we categorize them into proteases, esterases, carbohydrases, and so forth. However, if trypsin and carboxypeptidase are capable of cleaving ester bonds, and if this Cleavage proceeds faster than The breakdown of the corresponding peptides, we must abandon the notion that enzyme specificity is defined solely by the type of bond cleaved.

Similarly, we must discard the traditional division of proteolytic enzymes into endo- and exopeptidases. Until recently, it was presumed that exopeptidases act exclusively on peptides possessing a free amino or carboxyl group, which led to their further subdivision into aminopeptidases and Carboxypeptidases. Recently, however, it has been shown that leucine aminopeptidase is capable of hydrolyzing glycylleucinamide, even though the leucine residue in this substrate lacks a free amino group [26].

An examination of the structural formulas of the substrates acted upon by trypsin and carboxypeptidase (see above) makes it evident that the action of these enzymes depends primarily on the stereochemical configuration of the substrate's molecular groups, their shape, and their mutual arrangement. The Chemical Nature of the bonds being cleaved appears to be of secondary importance. This viewpoint is in full agreement with the proposition that enzyme-substrate binding occurs via interactions established between their complementary, shape-matched surfaces.