Salt-Activated Enzymes

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Cytochrome c can easily be extracted from tissue particles by dilute salt solutions. It was isolated by Keilin and Hartree in 1930 and shown to contain a porphyrin ring structure. In 1933 Zeilen and Reuter established that cytochrome c was a heme (iron-porphyrin) protein. Slightly different forms of cytochrome a were distinguished in yeast and bacteria by Keilin in 1934 and the different properties of cytochrome a and a3 by Tamiya et al. in 1937. The identity of cytochrome 03, the enzyme which activates oxygen with Warburg s atmungsferment, was proposed by Keilin in 1939. Cytochrome a/a3 was renamed cytochrome oxidase by Malcolm Dixon (1939). The oxidation route then offered was ... 

Various pancreatic enzymes hydrolyze lipids, including lipase with its auxiliary protein colipase (see p. 270), phospholipase A2, and sterol esterase. Bile salts activate the lipidcleaving enzymes through micelle formation (see below). 

Could this salt activation phenomenon be a result of relaxed diffusional limitations in a concentrated salt/enzyme formulation as compared to the salt-free preparation To answer this question, Bedell et al. [99] measured the initial rates of subtilisin Carlsberg-catalyzed transesterification of APEE with n-PrOH in hexane (Scheme 3.4) with two different enzyme preparations (Figure 3.8) (a) 98% (w/w)... 

These results were then correlated to the Jones-Dole coefficient to investigate the dependence of enzyme activation on the kosmotropicity of the salt in a solvent such as hexane. Specifically, plotting enzyme activity as a function of the difference in J DB coefficients of the cations and anions of the salts, resulted in a clear trend towards increased enzyme activity when the difference between the kosmotropicity of the anion and the chaotropicity of the cation was increased (Figure 3.10) [46]. These results were consistent with those of Ru et al. [33], in that enzyme activity in salt-activated preparations in hexane positively correlates with increased kosmotropicity on the anion. As a result of the elucidation of the influence of the kosmotropic/chaotropic properties of salts on enzyme function, the role of water... 

Solvent polarity is known to affect catalytic activity, yet consistent correlations between activity and solvent dielectric (e) have not been observed [12,102]. However, a striking correlation was found between the catalytic efficiency of salt-activated subtilisin Carlsberg and the mobility of water molecules (as determined using NMR relaxation techniques) associated with the enzyme in solvents of varying polarities (Figure 3.11) [103]. As the solvent polarity increased, the water mobility of the enzyme increased, yet the catalytic activity of the enzyme decreased. This is consistent with previous EPR and molecular dynamics (MD) studies, which indicated that enzyme flexibility increases with increasing solvent dielectric [104]. 

Eppler et al. [103] viewed these results as having a potential relationship to salt-activated enzyme preparations, particularly in relation to the mobility of enzyme-bound water. Specifically, the authors examined both water mobility [as measured by T2-derived correlation times, (tc)D20] and NaF-activated enzyme activity and observed a linear relationship. This suggests that the salt-activated enzymes contain a more mobile water population than salt-free enzymes, which facilitates a more aqueous-like local environment and dramatically increases enzyme activity through increased flexibility. Therefore, enzyme activation appears to correlate with the properties of enzyme-associated water. Once again, the physicochemical properties of water dictate enzyme structure, function, and dynamics. Hence, salt activation has proven to be a useful technique in activating enzymes for use in organic solvents and has provided a quantitative tool to better understand the role of water in enzymatic catalysis in dehydrated media. 

In addition to LPL, human milk contains a bile salts-activated lipase, which probably contributes to the metabolism of lipids by breast-fed babies who have limited pancreatic lipase activity. Bovine milk and milks from other dairy animals do not contain this enzyme. 

B. A. Bedell, V. V. Mozhaev, D. S. Clark, and J. S. Dordick, Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents, Biotechnol. Bioeng. 1998, 58, 654—657. 

D. S. Clark, 2001, Towards more active biocatalysts in organic media increasing the activity of salt-activated enzymes, Biotechnol. Bioeng. 75, 187-196. 

A stabilization model proposed for AMDH by Zaccai et al. (1989) accounts for all of the observations on the stability of this protein and on its solution structure. It is based on the fact that the AMDH solution particles are different in the different solvents in which the enzyme is active, and on the reasonable assumption that the bound water and salt molecules are not associated separately with the polypeptide but as hydrated salt ions. 

Inhibition. Since papain, ficin, and bromelain are all enzymes whose activity depends on a free SH group, it is to be expected that all thiol reagents act as inhibitors. Thus, a-halogen acids or amides and N-ethyl-maleimide irreversibly inhibit the thiol proteases. Heavy metal ions and organic mercurial salts inhibit in a fashion that can be reversed by low molecular weight thiols, particularly in the presence of EDTA which... 

Another aspect of heating soybeans in particular is the impact on the phospholipase enzyme. The phospholipase enzyme is activated at approximately 55°C and remains activated up to approximately 100°C. In this temperature range, and with sufficient exposed surface area and time, the phospholipase enzyme modifies a portion of the phospatides in the oil fraction by splitting off the non-fatty acid moiety (16). The resultant calcium and magnesium salts of phosphatidic acids that are formed tend to be more oil-soluble than water-soluble, thereby converting phospatides from a hydratable form to a nonhydratable form (16). This has a resultant impact on the quantities of acid, caustic and silica needed to reduce the phosphorus content of the soybean oil in the downstream degumming and refining unit operations. 

Human milk contains a bile salt-activated lipase (BAL). This enzyme helps infants in the digestion of milk fat (for a review, see Olivecrona... 

Pancreatic lipase, in the presence of bile salts and coUpase, acts at the oil-water interface of the triglyceride emulsion to produce fatty acids and 2-monoacylglycerols. Cohpase is secreted in pancreatic juice as an inactive proenzyme, which is converted to the active form by trypsin. Other significant enzymes involved in the breakdown of fats within the intestinal lumen are cholesterol ester hydrolase, phospholipase A, and a nonspecific bile salt-activated lipase. 

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