Subtilisin similarity

The active site of subtilisin is outside the carboxy ends of the central p strands analogous to the position of the binding sites in other a/p proteins as discussed in Chapter 4. Details of this active site are surprisingly similar to those of chymotrypsin, in spite of the completely different folds of the two enzymes (Figures 11.14 and 11.9). A catalytic triad is present that comprises residues Asp 32, His 64 and the reactive Ser 221. The negatively charged oxygen atom of the tetrahedral transition state binds in an oxyanion hole,... 

The single mutation Asp 32-Ala reduces the catalytic reaction rate by a factor of about lO compared with wild type. This rate reduction reflects the role of Asp 32 in stabilizing the positive charge that His 64 acquires in the transition state. A similar reduction of kcat and kcat/ m (2.5 x 10 ) is obtained for the single mutant Asn 155-Thr. Asn 155 provides one of the two hydrogen bonds to the substrate transition state in the oxyanion hole of subtilisin. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

FIGURE 9.4. The autocorrelation function of the time-dependent energy gap Q(t) = (e3(t) — 2(0) for the nucleophilic attack step in the catalytic reaction of subtilisin (heavy line) and for the corresponding reference reaction in solution (dotted line). These autocorrelation functions contain the dynamic effects on the rate constant. The similarity of the curves indicates that dynamic effects are not responsible for the large observed change in rate constant. The autocorrelation times, tq, obtained from this figure are 0.05 ps and 0.07ps, respectively, for the reaction in subtilisin and in water. 

Later on the crucial role played by the solvent was enlightened in the protease-catalyzed resolution of racemic amines [26]. As shown in Table 1.3, the ratio of the initial rates of acylation of the (S)- and the (Ji)-enantiomers or racemic a-methyl-benzylamine (9) varied from nearly 1 in toluene to 7.7 in 3-methyl-3-pentanol. Similarly, the same authors found a significant solvent effect for the subtilisin-catalyzed transesterification of racemic 1-phenylethanol (10) using vinyl butyrate as acyl donor (Table 1.4 [27]). 

KR was an alcalase subtilisin Carlsberg as the major enzyme component). To avoid racemization of the final product, they employed a mixture of 2-methyl-2-propanol/ H2O (19 1). Under these conditions, the product precipitated during the course of hydrolysis (Figure 4.26). A very similar DKR process was reported two years later by Parmar [52]. 

Proteins can be classed into groups based on their overall 3-D shapes, known as protein folds (O Figure 22-la). In general, proteins that have similar functions have similar folds. This means that if you are the proud parent of an unknown protein whose structure is solved, it may be possible to make educated guesses as to the function of the protein based on its overall fold. There are a number of well-known exceptions to this [notably, the serine protease family, subtilisin and trypsin/chymotrypsin (Hartley, 1979)], but the... 

An entirely different property of subtilisin was affected by substituting leucine at the 222 location. Native BPN is extremely sensitive to the presence of oxidation agents, showing rapid inactivation when incubated in the presence of 0.3% H2O2 (Figure 4). The Leu-222 variant, in contrast, was found to be totally stable under the same oxidation conditions. The data clearly show that single amino acid alterations can have dramatic effects upon the activity of the enzyme. Similarly, other changes have been shown to affect catalytic properties, substrate specificities and thermostability (7,2,9). 

Recently, improved hydrophilicity and dyeability with acid and disperse dyes of nylon 6 fibres after treatment with protease (subtilisin) was reported [31]. Similarly, various proteases were used for surface hydrolysis of nylon 6,6 fibres, leading to... 

Early reports on the effects of the choice of solvent on enzymatic enantioselectivity showed that substantial changes may be observed. For the transesterification reaction of sec-phenethyl alcohol with vinyl butyrate catalyzed by subtilisin Carlsberg, a 20-fold increase in the E-value was reported when the medium was changed from acetonitrile to dioxane [59]. Similar changes were recorded for the prochiral selectivity of Pseudomonas sp. lipase in the hydrolysis of 2-substituted... 

It was found that MpSCD is able to restore dehydration-induced structural perturbations to a certain extent. Results were interpreted to show that enantioselectivity and structural intactness in the various solvents investigated were clearly related. Increased enzyme activity, in contrast, is mainly caused by increased structural flexibility of subtilisin in the solvents by M(iCD. Similar results have been reported for subtilisin Carlsberg [124]. 

Naturally occurring Upases are (R)-selective for alcohols according to Kazlauskas rule [58, 59]. Thus, DKR of alcohols employing lipases can only be used to transform the racemic alcohol into the (R)-acetate. Serine proteases, a sub-class of hydrolases, are known to catalyze transesterifications similar to those catalyzed by lipases, but, interestingly, often with reversed enantioselectivity. Proteases are less thermostable enzymes, and for this reason only metal complexes that racemize secondary alcohols at ambient temperature can be employed for efficient (S)-selective DKR of sec-alcohols. Ruthenium complexes 2 and 3 have been combined with subtilisin Carlsberg, affording a method for the synthesis of... 

Alternatively, the enzyme can be modified such that it dissolves in a hydrophobic ionic liquid with retention of activity. This approach was demonstrated with cyt c, which, when covalently modified with polyethylene glycol (PEG), dissolved in [EMIm][ Tf2N] with retention of activity. The best results were obtained when the molecular weight of the polymer chain was >2000 [88]. Similarly, a copolymer of PEG and maleic anhydride solubilized subtilisin in [EMIm][NTf2] and a range of similar ionic liquids with good retention of activity and operational stability [89, 90]. 

---