Enzymes subtilisin

In order to develop a host strain for production of a variety of subtilisin enzymes, it was first necessary to delete the endogenous alkaline and neutral proteases (72,76). The strain was then constmcted to contain the optimal combination of subtilisin regulatory genes which were compatible with the proposed fermentation and recovery process. Once this strain had been produced, the recombinant enzyme... 

Parvinzadeh M (2009) A new approach to improve dyeability of nylon 6 fibre using a subtilisin enzyme. Coloration Technol 125 228-233... 

As for subtilisin BPN, the first attempt at molecular modeling of disulfide mutants was performed with computer graphics using coordinates from the crystal structure of the enzyme,10-121 but increasing enzyme stability was unsuccessful. We have studied a thermostable subtilisin-type protease, aqualysin I, and the introduction site of a disulfide bond was chosen on structural homology between aqualysin I and subtilisin E. Here we describe a successful study to increase the stability of subtilisin E and others done for subtilisin enzymes. 

Bott, R., and Betzel, C. (1996). Subtilisin Enzymes, Plenum Press, New York. 

Klein AE, Freiberg J, Same S et al. (1989) Rapid colorimetric determination of activity of subtilisin enzymes in cleaning products. Assoc Off Anal Chem 72(6) 881-882 Klibanov AM (1977) A new approach to preparative enzymatic synthesis. Biotechnol Bioeng 28 417 21... 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

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,... 

Figure 11.14 Schematic diagram of the active site of subtilisin. A region (residues 42-45) of a bound polypeptide inhibitor, eglin, is shown in red. The four essential features of the active site— the catalytic triad, the oxyanion hole, the specificity pocket, and the region for nonspecific binding of substrate—are highlighted in yellow. Important hydrogen bonds between enzyme and inhibitor are striped. This figure should be compared to Figure 11.9, which shows the same features for chymotrypsin. (Adapted from W. Bode et al., EMBO /.

All the four essential features of the active site of chymotrypsin are thus also present in subtilisin. Furthermore, these features are spatially arranged in the same way in the two enzymes, even though different framework structures bring different loop regions into position in the active site. This is a classical example of convergent evolution at the molecular level. 

By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

The subtilisin mutants described here illustrate the power of protein engineering as a tool to allow us to identify the specific roles of side chains in the catalytic mechanisms of enzymes. In Chapter 17 we shall discuss the utility of protein engineering in other contexts, such as design of novel proteins and the elucidation of the energetics of ligand binding to proteins. 

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. 

Stibine, see Antimony hydride Stoddard solvent Strontium chromate Strychnine Styrene, monomer Subtilisins (proteolitic enzymes as 100% pure crystalline enzyme)... 

Furin, also known as paired basic amino-acid-cleaving enzyme (PACE), is a membrane bound subtilisin-like serine protease of the irons Golgi compartment. It is ubiquitously expressed and mediates processing of many protein precursors at Arg-X-Lys/Arg-Arg sites. 

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

FIGURE 7.9. The Asn-155- Ala mutation in subtilisin involves deletion of a hydrogen bond between the enzyme and the oxyanion transition state. 

An examination of the autocorrelation function (0(0) <2(0) annucleophilic attack step in the catalytic reaction of subtilisin is presented in Fig. 9.4. As seen from the figure, the relaxation times for the enzymatic reaction and the corresponding reference reaction in solution are not different in a fundamental way and the preexponential factor t 1 is between 1012 and 1013 sec-1 in both cases. As long as this is the case, it is hard to see how enzymes can use dynamical effects as a major catalytic factor. 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

SCF, see Self-consistent field treatment (SCF) Schroedinger equation, 2,4,74 Secular equations, 6,10, 52 solution by matrix diagonalization, 11 computer program for, 31-33 Self-consistent field treatment (SCF), of molecular orbitals, 28 Serine, structure of, 110 Serine proteases, 170-188. See also Subtilisin Trypsin enzyme family comparison of mechanisms for, 182-184, 183... 

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

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]. 

The KR of secondary alcohols by some hydrolytic enzymes has been well known. The combinations of these hydrolytic enzymes with racemization catalysts have been explored as the catalysts for the efficient DKR of the secondary alcohols. Up to now, lipase and subtilisin have been employed, respectively, as the R- and 5-selective resolution enzymes in combination with metal catalysts (Scheme 2). 

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