Latent enzymes

Miller, B.G. and Raines, R.T. (2004) Identifying latent enzyme activities substrate ambiguity within modem bacterial sugar kinases. Biochemistry, 43, 6387-6392. 

The elimination or inactivation of enzymes used to treat proteins is a critical problem once the desired modification in functionality is achieved. In many instances, product inhibition or self destruction does not occur as noted above for fish protein concentrate. As stated by Puski (20), if heat inactivation is used, the proteins may be denatureT"and revert to insoluble forms. Washing out the enzyme at its isoelectric point would also remove a portion of the protein which is solubilized by the enzyme. Inactivation of enzymes by chemical means may also cause significant changes in the protein. Thus, while desired functional modifications of food ingredients may be obtained through enzyme treatment, the problem of latent enzyme activity in food formulations must be addressed. 

Latent forms of MMPs can be activated by mechanisms which cause the dissociation of the intramolecular complex between a particular cysteine residue and the required zinc metal ligand (a complex that blocks the active site) [47], This occurs because the cysteine of the latent enzyme is coordinated to the active site in a particular way that blocks the MMP active site. Collectively, the activation of MMPs occurs through a process which has been termed the cysteine-switch . Activators of the MMPs include proteases (e.g. plasmin), conformational perturbants (SDS, NaSCN), heavy metals and organomercurials (e.g. Au(I) compounds, Hg(II)), oxidants (e.g. OC1-), disulfide compounds (e.g. GSSG) and sulfhydryl alkylating agents (e.g. V-ethylmaleimide) [47 and refs, therein]. 

Mushroom tyrosinase was extracted as described by Ingebrigtsen and Flurkey (J. Food Sci., in press). Tyrosinase activity was monitored using either catechol, dopa or tyrosine as the substrates. All assays were carried out in the presence and absence of 0.1% SDS (w/v) to detect active and latent enzyme activities. The catechol oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 10 mM catechol and the absorbance monitored at 410 nm (25-26). The dopa oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 5 mM L-dopa and the absorbance monitored at 475 nm. The tyrosine hydroxylase activity of tyrosinase was assayed in 33 mM phosphate (pH 6.0) containing 0.33 mM L-tyrosine and the absorbance monitored at 280 nm. Protein content was determined by the method of Lowry et al. (26). 

Figure 4. Clear bars represent active tyrosinase in four developmental stages in each of three different breaks. Hatched bars represent active and latent enzyme using dopa as the substrate. (Reproduced with permission from ret 35. Copyright 1989 J. Food ScL)...

An a-D-mannosidase (acid pH optimum) that is Zn " -dependent and three other a-D-mannosidase activities (neutral pH optima) have been found in the tissues of rats. The enzymes most active at a neutral pH value were inhibited by heavy-metal ions and were stabilized, to some extent, by thiols. The reversible effects of Fe , Co and Mn ions and H edta on the activity of these enzymes were investigated. Tissue preparations varied widely in the levels of the enzymes most active at pH 6.5, although latent enzymic activity was released on incubation with activating metal ions. 

Several enzymes are not detected when fresh mitochondria are assayed, but are detected when the preparations are aged, warmed, frozen, or otherwise maltreated. These treatments, that cause loss of some activities, permit measurement of latent enzymes, including ATPase, glutamic dehydrogenase, and DPN cytochrome reductase. Some of the latent enzymes are readily solubilized by disruption of the mitochondria, while others remain associated with large sedimentable fragments. ... 

Yeasi eniymes The pmduclion of carbon dioxide in yeast breads is catalyzed by many dil latent enzymes found in the yeast. 

Only thiosulfate and thiosulfonates, but not (8-mercaptopyruvate are substrates of rhodanese. Sulfite can replace CN as the S acceptor. The physiological role of rhodanese is unknown, its concentration increases in the fetus until birth, but remains constant in the mother during pregnancy (91). Rhodanese is a latent enzyme in isolated mitochondria and becomes activated upon structural derangement of the mitochondrion caused by aging or hypotonic salt solution (98). 

The second point concerns the exchange of the nucleotides bound on CFj. On isolated CF], the latent enzyme, the binding sites fall into two different categories. 

The efficiency of inactivation by covalent bond formation vs release of the reactive species into solution has been described by its partition ratio. The most efficient inactivators have catalytic partition ratios of 0, in which case each inhibitor molecule leads to inactivation of the enzyme. To this date, many of these inhibitors have been designed, and alternative names like suicide substrate, Trojan Horse inactivator, enzyme induced inactivator, inhibitor, and latent inactivator have been used for this class of inhibitors. A number of comprehensive reviews are available (26—32). 

The matrix metalloproteinases are inhibited by specific endogenous tissue inhibitor of metalloproteinases (TIMPs), which comprise a family of four protease inhibitors TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Overall, all MMPs are inhibited by TIMPs once they are activated but the gelatinases (MMP-2 and MMP-9) can form complexes with TIMPs when the enzymes are in the latent form. 

Facilitates the breakdown of protein in the muscle, leading to increased plasma amino acid levels. Increases activity of enzymes necessary for glucogenesis producing hyperglycemia, which can aggravate diabetes, precipitate latent diabetes, and cause insulin resistance... 

Mineral dust-induced ROMs contributes to pulmonary fibrosis, malignancy, hypersensitivity and emphysema (Doelman etctl., 1990 Kamp etui., 1992). The involvement of ROMs in pulmonary fibrotic reactions is indicated by the participation of PMN oxidants in the autoactivation of latent coUagenase (Weiss et al., 1985). Prolyl hydroxylase, a key enzyme in collagen fibril formation, has been shown to be dependent on the reaction of superoxide with prolyl residues (Myllyla et al., 1979). 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

The studies described above show that a quinone methide or its aza-analogue quinonimine methide incorporated as a latent electrophilic species into a cyclic lactone or lactam precursor can modify a second nucleophilic residue within the enzyme active site after formation of the acyl-enzyme. Very efficient suicide... 

The matrix metalloprotease (MMP) family of zinc hydrolases are thought to play important roles in extracellular tissue remodeling in angiogenesis and other normal physiological processes, in some inflammatory processes and in metastatic processes in cancer. Like the zinc carboxypeptidases, the MMPs also utilize a zinc-coordinated water molecule to initiate attack on the scissile amide bond of protein substrates. These enzymes are synthesized by the ribosome in a latent form composed of a catalytic domain and an N-terminal extension, referred to as the prodomain the latent, or inactive form of the enzyme is referred to as a zymogen or... 

The temperate virus does not exist in its mature, infectious state inside the cell, but rather in a latent form, called the provirus or prophage state. In considering virulent viruses we learned that the DNA of the virulent virus contains information for the synthesis of a number of enzymes and other proteins essential to virus reproduction. The prophage of the temperate virus carries similar information, but in the lysogenic cell this information remains dormant because the expression of the virus genes is blocked through the action of a specific repressor coded for by the virus. As a result of a genetic switch, the repressor is inactivated, virus reproduction occurs, the cell lyses, and virus particles are released. 

Over the past years it has become apparent that the cell type is an important determinant of the extent of oxidative stress that may occur. Both the latent activities of cytoprotective enzymes in specific cell types, as well as the ability of the cell to respond rapidly to an oxidative insult by the upregulation of such enzymes, will be important predeterminants of the fate of the cell. Table 10.1 shows the concentrations of both antioxidants and cytoprotective enzymes in a variety of tissues. While the liver is well provided with antioxidant protection, the brain has very low levels, so the ability to respond rapidly to an oxidative insult by upregulation of gene transcription and translation will be an important determinant of survival or death. Cells such as hepatocytes have high levels of expression and... 

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