Subtilisin active site studies

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

The closest organic specie to the inorganic boric acid are the boronic acids generally described as R-B(OH)2. Boronic acids have been shown to act as inhibitors of the subtilisins. X-ray crystallographic studies of phenylboronic acid and phenyl-ethyl-boronic acid adducts with Subtilisin Novo have shown that they contain a covalent bond between the oxygen atom of the catalytic serine of the enzyme and the inhibitor boron atom (Matthews et al, 1975 and Lindquist Terry, 1974). The boron atom is co-ordinated tetrahedrally in the enzyme inhibitor complex. It is likely that boric acid itself interacts with the active site of the subtilisins in the same manner. 

St Leger et al. (1987b) characterized two subtilisin-like proteases (chymoelastases) and three tiypsin-like proteases from M. anisopliae. A subtilisin-like protease (Prl pl=10.3, Mw=25 kDa) and a trypsin-like protease (Pr2 pl=4.42, Mw=28.5 kDa) were purified to homogeneity. Inhibition studies have revealed that both enzymes possess essential Ser and His residues in the active site. Prl exhibited higher activity to locust cuticle than Pr2 and it showed activity to elastin as well. 

The subtilisins are a large family of serine proteases that have been extensively studied because of their importance in the detergent industry. The retain a common conserved fold and identical active site residues (Fig. 10). Two conserved calcium-binding sites are present in all subtilisins, and additional sites may be present in subtilisins from different organisms (Siezen and Leunissen, 1997). 

The techniques of molecular biology discussed in Chapter 6 have permitted detailed examination of the catalytic triad. In particular, site-directed mutagenesis has been used to test the contribution of individual amino acid residues to the catalytic power of an enzyme. Subtilisin has been extensively studied by this method. Each of the residues within the catalytic triad, consisting of aspartic acid 32, histidine 64, and serine 221, has been individually converted into alanine, and the ability of each mutant enzyme to cleave a model substrate has been examined (Figure 9.16). As expected, the conversion of active-site serine 221 into alanine dramatically reduced catalytic power the value of k fell to less than one-millionth of its value for the wild-type enzyme. The value of. Sf was essentially unchanged its increase by no more than a factor of two indicated that substrate binding is not significantly affected. The mutation of histidine 64 to alanine... 

The earliest observation that implied evolutionary links between all lipases was that of the consensus pentapeptide G-X-S-X-G, subsequently shown to contain the nucleophilic serine. The apparent similarity of this sequence to that found around the active serine in the chy-motrypsin and subtilisin families of serine proteinases prompted a number of authors to infer an evolutionary relationship between the three families. Further evidence in support of such a link came from secondary structure prediction studies indicating that the nucleophilic serine in a lipase is most likely within a /3 turn, structurally reminiscent of proteinases (Reddy et ai, 1986). In fact, one of the commonly used phrases found in introductions to many papers dealing directly or indirectly with lipases refers to the consensus G-X-S-X-G pentapeptide found in the active site of all serine proteinases and esterases. We now know that the implication that homology and/or structural similarities exist between the enzymes belonging to these diverse groups is incorrect. The matter has been dealt with in the literature (Derewenda and Derewenda, 1991 Liao et ai, 1992), but it seems appropriate to review some of the conclusions. 

Support for an even more remote electrostatic effect on the mechanism is supplied by the studies of Jackson and Fersht (1993). They mutated charged residues on the surface of Subtilisin BPN, that are 13-15A from the active site, to either neutral or oppositely charged residues. The effect of those mutations on the inhibition constant, Ki, of a trifluoromethyl ketone, was compared for wt and mutated subtilisin. The mutations were Asp-36 (located on a surface loop outside the active site cleft and separated from His-64 by about 15-16A) to Gin, and of Asp-99 (about 12-13A from His-64) to Ser and to Lys. The active site of subtilisin includes Ser-221, His-64 and Asp-32. 

The approach of the substrate towards the active site of SP was studied with electrostatic field potentials (ESP) by Lamotte-Brasseur et al. (1990). They examined the force exerted by the active sites of the serine proteinases alpha-chymotrypsin and subtilisin on an approaching substrate. About 20 residues from each of these enzymes were employed to construct electrostatic potential maps, using point charges derived from CNDO calculations (Pople and Beveridge, 1970) with e =l.Net charges were obtained from a mulliken analysis... 

The construction of enzymes with new substrate specificities is now a realistic goal, and some novel approaches have been presented. For example, removal of an active-site histidine by the His-64 Ala mutation in subtilisin results in an enzyme with markedly reduced activity, but one which can be enhanced 400-fold with substrates containing histidine at the PI site (759). Apparently, the substrate histidine assists catalysis by partially compensating for the role of the lost active-site His-64. In a similar study, mutation of Lys-258 to Ala in aspartate aminotransferase produces an enzyme whose activity can be restored by exogenous amines (140). 

Recent data of Fraser, Corstorphine, and Zammit using trypsin and proteinase K treatment of intact mitochondria and outer-membrane-ruptured mitochondria suggest that both the active site and the malonyl-CoA binding site are exposed on the cytosolic side of the membrane and that CPT-I has two transmembrane domains. Thus, we have conducted additional studies of the topology of CPT-I using intact hepatic mitochondria and isolated mitochondrial outer membranes with Nagarse (subtilisin BPN ), papain, and trypsin using a variety of incubation conditions. 

Studies of y-chymotrypsin by Segal et al and of subtilisin by Kraut et al. with chloromethyl ketone analogues of good phenylalanine polypeptide substrates have indicated further similarities in substrate binding and specificity of these enzymes. It has previously been reported that both enzymes contain a serine at the active site which is acylated by ester substrates, a histidine hydrogen-bonded to this serine which is alkylated by active-site directed halogenomethyl ketones, and an aspartate which is buried and hydrogen-bonded not only to the active-site histidine but also in each case to a further serine. 

It has been suggested that the active sites in proteins are better conserved than the overall fold [27]. If so, then one should be able to identify not only distant ancestors with the same global fold and same biochemical activity, but also proteins with similar functions but different global folds. Nussinov and coworkers empirically demonstrated that the active sites of eukaryotic serine proteases, subtilisins, and sulfhydryl proteases exhibit similar structural motifs [216]. Furthermore, in a recent modeling study of S. cerevisiae proteins, active... 

A new sorbent for the biospecific chromatography of serine proteinases has been obtained via attachment of p-(reversible inhibition by organoboronic acids has been used to study the active site topography of subtilisin. ... 

To assess the effect of protein binding on catalysis, we compared the initial rates of [1] oxidation by t-butylhydroperoxide in the presence of selenolsubtilisin and diphenyldiselenide, a nonenzymatic model compound. At low substrate concentrations the enzymatic reaction is roughly 70,000 times faster than the selenocystein catalyzed process. Clearly, the redox activity of the selenium group is considerably increased in the active site of subtilisin. Further characterization of the redox chemistry of selenolsubtilisin is in progress. We believe that the information gained in this study will provide a better understanding of how natural glutathione peroxidase works. 

The new generations of experiments are aimed at linking dynamical studies of these and other processes to the function. We have already begun research in this direction. In a recent publication [9] we reported studies of the femtosecond dynamics of an RNA-protein complex and then compared the results with those obtained for in vivo (E. Coli) transcription anti-termination activities. In two other studies we measured the activity of the protein Subtilisin Carlsberg, discussed above, to a substrate, and the role of hydration in interfacial binding and function of bovine pancreatic phospholipase at a substrate site. The goal in all these studies is to relate structures to the dynamics and hopefully to key features of the (complex ) function. 

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