Pepstatin inhibition kinetics

While the inhibition kinetics of smaller statyl compounds are fairly straightforward, the kinetics study of statyl-containing tetrapeptide, N-acetyl-Val-Sta-Ala-Sta, and pepstatin itself are much more complicated. First, it is more difficult to measure of tight binding inhibitors. Moreover, if the statyl residues were the source of inhibitory potency , the presence of two statyl residues in each molecule of inhibitor casts further doubt on the theoretical validity of kinetic parameters. 

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

Since pepstatin binds so tightly to pepsin, it was not suitable for kinetic studies of the inhibition of pepsinogen activation. Consequently, we chose to observe the activation of pepsinogen in the presence of various concentrations of globin, the protein moiety from hemoglobin. For these assays, pepsinogen was allowed to activate at pH 2 in the presence of a known concentration of globin. 

Since pepstatin is an extremely potent inhibitor, the study of the kinetics of its inhibition is difficult. The kinetic approach does appear to be warranted, however, because pepstatin, while a strong-binding inhibitor, is not an irreversible inactivator. This... 

To further study the mode of inhibition of pepsin and other acid proteases by pepstatin, we tried to produce various pepstatin fragments and measure their kinetics of inhibition. These pepstatin fragments are produced by three methods (a) complete acid hydrolysis yields free statine and other free amino acids (b) partial acid hydrolysis yields a mixture of peptides and (c) enzyme digestion with a-lytic protease yields the tetrapeptide, valyl-statyl-alanyl-statine. Purification by high-voltage paper electrophoresis from the above mixtures gives four statine-containing products in sufficient yield to allow further study statine, alanyl-statine, valyl-statine, and valyl-statyl-alanyl-statine. The isolated products are acetylated and quantified, and the completeness of their acetylation is determined prior to the initiation of inhibition studies (10). 

An interesting question can thus be raised. If pepstatin is a transition state inhibitor by virtue of the structure of statine, why then is there a thousandfold difference in values observed for various acid proteases Since there is no extensive kinetic data available for the inhibition of a variety of acid proteases by smaller statyl compounds, we can only speculate as to the answers. 

To study its mode of inhibition, we prepared several derivatives and measured their kinetics of inhibition. Both N-acetyl-statine and N-acetyl-alanyl-statine are competitive inhibitors for pepsin with values of 1.2 X lO M and 5.65 x 10 M, respectively. The value for N-acetyl-valyl-statine is 4.8 x 10 M. These statyl derivatives, therefore, are very strong inhibitors. The value for N-acetyl-statine is 600-fold smaller than that of its structural analog N-acetyl-leucine. The derivative which contains two statyl residues in a tetrapeptide exhibits inhibitory properties which approach those of pepstatin itself. Other acid proteases, human pepsin, human gastricsin, renin, cathepsin D, the acid protease from R. chinensis and bovine chymosin, also are inhibited by pepstatin and its derivatives. We suggest that the statyl residue is responsible for the unusual inhibitory capability of pepstatin and that statine is an analog of the previously proposed transition state for catalysis by pepsin and other acid proteases. 

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