Enzymes proteinase

Melrose (1971) reviewed the instances in which direct comparisons of the stabilities of native and immobilized enzyme pairs were reported In 30 cases the insoluble enzyme was more stable, in 8 the soluble, whilst there was little difference in the remaining 12 cases. Immobilization does not lead necessarily to stabilization ho ever, this is mainly trae in the immobilization of self-destructing enzymes (proteinases). A comprehensive review on enzyme stabilization by immobilization has been written byKlibanov (1979). 

In addition to carboxylesterases, another hydrolyzing enzyme, proteinase, is also involved in resistance. Oppert et al. (1997) showed that two Bt-resistant strains of the Indian meal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins as compared with susceptible strains. The absence of this enzyme is genetically linked to larval susceptibility to the toxin. 

Telastaside (213) showed dose-dependent biphasic immunomodulatory responses (25). Its effects suggested inhibition or augmentation of enzyme (proteinase) and membrane integrity, as revealed from the viability or inhibition of growth of both normal and tumor cells (164). 

From Sephadex G-10 and G-15 gel filtration, the molecular weight of CF was calculated as about 700 daltons (16). To obtain further information about the chemical nature of CF, the active CF fraction, isolated after Sephadex G-10 gel filtration, was treated with different enzymes. Enzymes proteinase K, cellulase, a and / amylase did not reduce the CF activity (data not shown). 

A common method for RNA isolation is the proteinase K method. In this method, the cells are lysed by incubation in a hypotonic solution followed by centrifugation to remove DNA and cell debris. Treatment with the proteolytic enzyme proteinase K leads to the dissociation of RNA-protein complexes and the digestion of the proteins. The digestion products are then removed by phenol/chloroform extraction and the RNA in the remaining aqueous solution is precipitated using ethanol. 

For this study we chose to modify the activity, pH optimum and thermostability of the enzyme proteinase K from Tritirachium album. Hydrolytic enzymes such as proteases and lipases are finding many applications in polymer synthesis and degradation (74-27). The activity and physical properties of these enzymes determine whether or not they will make suitable biocatalysts for polymer modifrcation. 

The pancreas is the most important site of production of digestive enzymes proteinases, peptidases, lipases, nucleases, etc. (cf. Chapt. VII-9, VIII-1, and XII-2). The amount of enzymes produced has been estimated to be 15-30 gm per day, which is considerable. The pancreatic juice, itself alkaline, enters the intestine and first neutralizes the acidity coming from the stomach. When acidic food pulp passes the pylorus, the latter stimulates the pancreas to elaborate more of its digestive enzymes. The secretion of pancreatic juice is regulated by tissue hormones ( secretin, Chapt. XX-12). 

Hydrolases. Enzymes catalysing the hydrolytic cleavage ofC —O, C —N and C —C bonds. The systematic name always includes hydrolase but the recommended name is often formed by the addition of ase to the substrate. Examples are esterases, glucosidases, peptidases, proteinases, phospholipases. Other bonds may be cleaved besides those cited, e.g. during the action of sulphatases and phosphatases. 

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

In this chapter we shall illustrate some fundamental aspects of enzyme catalysis using as an example the serine proteinases, a group of enzymes that hydrolyze peptide bonds in proteins. We also examine how the transition state is stabilized in this particular case. 

The serine proteinases all have the same substrate, namely, polypeptide chains of proteins. However, different members of the family preferentially cleave polypeptide chains at sites adjacent to different amino acid residues. The structural basis for this preference lies in the side chains that line the substrate specificity pocket in the different enzymes. 

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

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. 

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. 

Peptidases are enzymes that catalyse the hydrolysis of peptide bonds - the bonds between amino acids that are found in peptides and proteins. The terms protease , proteinase and proteolytic enzyme are synonymous, but strictly speaking can only be applied to peptidases that hydrolase bonds in proteins. Because there are many peptidases that act only on peptides, the term peptidase is recommended. Peptidases are included in subclass 3.4 of enzyme nomenclature [1,5]. 

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