Pepsin catalysis

In 1878 the term enzyme, Greek for "in yeast," was proposed (8). It was reasoned that chemical compounds capable of catalysis, ie, ptyalin (amylase from sahva), pepsin, and others, should not be called ferments, as this term was already in use for yeast cells and other organisms. However, proof was not given for the actual existence of enzymes. EinaHy, in 1897, it was demonstrated that ceU-free yeast extract ("zymase") could convert glucose into ethanol and carbon dioxide in exactiy the same way as viable yeast cells. It took some time before these experiments and deductions were completely understood and accepted by the scientific community. 

This conclusion was verified by a C NMR experiment carried out in 2h2 8o which gave a 0.05 ppm upfield shift in the resonance for the C-3 carbon relative to the carbon resonance in 2H2 0. The upfield shift in the carbon resonance establishes that the oxygen nucleophile that adds to the C-3 carbonyl group when 6 binds to pepsin must come from water. These labeling results are not consistent with the addition of the Asp-32 carboxyl group to the carbonyl group to form a covalent tetrahedral species as would occur during nucleophilic catalysis. 

These data unambiguously establish that a gem diol species is formed in the active site of pepsin when the ketone analogs 6 and 10 are added to the aspartyl protease as shown in Figure 6 and exclude the formation of a covalent intermediate. Our data strongly support the general acid-general base catalysis mechanism for aspartyl proteases that is illustrated schematically in Figure 1. 

The present study indicates that the extracellular enzyme, pepsin, exhibits striking differences from its mammalian homologue with respect to optimum pH, Ea for catalysis, thermal stability, and substrate affinity. These data are interesting from the viewpoint of biological adaption at low temperatures, but they also provide some substance to our contention that enzymes from fish plant wastes can have sufficiently unique properties to justify their use over conventional sources of enzymes used as food-processing aids. The relatively low Eas for protein hydrolysis by fish pepsins indicate they may be especially useful for protein modifications at low temperatures. Alternatively, the poor thermal stability of the fish pepsins studied indicate that the enzymes can be inactivated by relatively mild blanching temperatures. The reality of this concept will have to await studies where the pepsins are used as food-processing aids. Such studies are currently underway in our laboratory. 

Data presented suggest that acid-base catalysis is of major importance in the hydrolytic action of pepsin. The pK of the beat carboxyl group of aspartate is around 4 in the free acid and may very well be lower in the protein molecule due to neighboring group effects. A pH optimum near the pK of aspartic acid suggests that the ability of aspartate to accept and donate protons is of major importance in its mode of action. 

Data is accumulating for pepsin and HIV protease to suggest that they may catalyze their reactions through a mechanism in which release of products leaves the enzyme in a conformation which must undergo isomerization before another round of catalysis can commence [65]. More work is needed here to eliminate other mechanistic possibilities. 

The term catalysis was applied to the biological phenomena listed above and to several other reactions discovered in the next several years. The active component of bitter almonds was named emulsin in 1837. Other carbohydrate-splitting activities (ptyalin in saliva, amylase in malt) had already been described. Pepsin and trypsin, protein-digesting agents from the stomach and pancreas, were also discovered during this period. The activities of these materials were contrasted with the materials responsible for fermentation. Early theories of Willis and of Stahl to explain fermentation as a disruption caused by violent motion of... 

Under acidic conditions, pepsinogen is converted to pepsin by autocatalytic (i.e., intramolecular) catalysis, which results in loss of an additional NHz-terminal sequence. This conversion occurs slowly at pH values of 5 to 6 but very rapidly at pH 2. One of the NHz-terminal peptides released during the conversion possesses antipepsin activity. This peptide (MW 3,200) and its analogs complex with pepsin at pH values of 5.0 to 6.0 to inhibit catalytic activity. However, at lower pH, the inhibitor dissociates and is digested by pepsin. Thus, it seems unlikely that the inhibitor serves any significant physiologic function, at least in the adult stomach. 

A cartoon depicting the ligands and the transduction mechanisms activating the chief cell to secrete pepsinogen. Under acidic conditions, cleavage of the N-terminal sequence of pepsinogen results in autocatalytic catalysis and activation to pepsin. 

The acid-protease from Rhizopus chinensis was first isolated by Fukumoto, Tsuru, and Yamamoto (1). Its optimum pH for catalytic activity was shown to be between 2.9 and 3.3 (1). Although its complete sequence has not yet been determined, some limited sequence data are available - particularly that of the 39 amino-terminal residues (2,3) and of the residues in the immediate vicinity of the catalytically active aspartic acid residues (4,5). These data show that this enzyme has substantial sequence homology with porcine pepsin. Investigations of the kinetics of catalysis (6,7) have led to proposals of an extended subsite specificity. 

Whereas the acyl dipeptide substrates of pepsin were resistant to acid proteinases such as cathepsin D (25,26), several of the more sensitive cationic substrates were cleaved by these and other members of this family of proteinases (27,28). The apparent differences in the specificity of the action of these enzymes have been shown to be a consequence, in large part, of the contribution made by secondary enzyme-substrate interactions to catalysis (29). 

If a covalently-bound amino-enzyme is indeed an intermediate in pepsin catalysis, it would be expected that a series of substrates of the type A-Phe-Trp or A-Phe(4N02)-Tyr, where the nature of the A group is varied, would yield the same intermediate (E-Trp or E-Tyr). It has been shown, however, that the ratio of labeled Ac-Phe-Trp formed in the presence of l C-labeled Ac-Phe to the Tryptophan formed by hydrolysis of A-Phe-Trp is not constant, and the substrate that is hydrolyzed most rapidly (A = Z-Ala-His) gave no detectable transpeptidation under the conditions of the experiments (52). Similarly, with substrates of the type A-Phe(4N02)-Tyr (A = Z, Z-Gly, Z-Gly-Gly), the importance of secondary interactions is clearly evident, and the increased is correlated with the decrease in the... 

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