Specificity and Mechanism of Pepsin Action on Synthetic Substrates

  • Joseph S. Fruton
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 92)

Abstract

Studies on the specificity and mechanism of pepsin action have involved the use of several types of synthetic substrates. In the early work, substrates related to Z-Glu-Tyr (1) or Ac-Phe-Tyr (2) were largely employed; for a review, see (3). In particular, much use had been made of acyl dipeptides of the type A-X-Y, where X and Y are aromatic L-amino acid residues forming the sensitive peptide bond. Because the pKa of the carboxyl group falls in the pH range of pepsin activity, the pH dependence of the kinetic parameters is a function of the ionization of prototrophic groups in both the enzyme and the substrate. The methyl or ethyl esters, or amides, of acyl dipeptides have also been used extensively, but because of their limited solubility in aqueous solution, variable amounts of organic solvents had to be added. Such solvents, even in relatively low concentration, markedly inhibit pepsin action (4,5). To obviate this difficulty, another type of pepsin substrate was introduced, in which a cationic group (the imidazolium group of a His residue, the α-ammonium group, a pyridinium group, or a morpholinium group) is present. By means of such substrates, in particular of the general structure Z-His-X-Y-OMe, the primary specificity of pepsin was defined as a preference for hydrophobic L-amino acid residues in both the X- and Y- positions (10); substitution of either Phe residue of Z-His-Phe-Phe-OMe by its D-enatiomer renders the X-Y bond resistant to pepsin action (6). The favorable effect of an aromatic and planar substituent at the β-carbon of the X and Y residues was emphasized by the finding that when X is β-cyclohexyl-L-alanyl, the value of kcat is much lower than that found for the corresponding substrate in which X or Y = Phe, and is similar to that for substrates in which the X- or Y-position is occupied by an aliphatic amino acid residue larger than Ala (Nva, Nle, Leu, Met). Apparently, the side chains of these amino acids can interact with a portion of the enzymic region that binds planar aromatic groups. It was also shown that the replacement of Phe in the X-position by Val or Ile rendered the X-Y bond resistant to pepsin action than when X = Gly, indicating that when the X-position is occupied by a residue that is branched at the β-carbon, one of the catalytic groups of pepsin may be prevented from attacking the carbonyl group of the sensitive bond. Moreover, the importance of the β-methylene group as a structural element of the X-Y unit was underlined by the finding that replacement of either Phe residue of Z-His-Phe-Phe-OMe by a L-Phenylglycyl residue also rendered the X-Y bond resistant to pepsin action (11).

Keywords

Hydrolysis Amide Tryptophan Benzyl Methoxy 

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Copyright information

© Springer Science+Business Media New York 1977

Authors and Affiliations

  • Joseph S. Fruton
    • 1
  1. 1.Kline Biology TowerYale UniversityNew HavenUSA

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