Proteins by enzymes

The above method, due to Sorensen, is of great importance in following the course of hydrolysis of proteins by enzymes (p. 516). For example, if the protein and its hydrolysis are represented thus ... 

Identification of the Isolate. Growth of HIV isolates in cell culture is monitored once or twice weekly by measuring reverse transcriptase (RT) activity or the production of viral p 24 core protein by enzyme immunoassay of the culture medium. Most laboratories have established cutoff values for each assay and also monitor rises in RT activity or antigen level. 

Proteolysis of peptides and proteins by enzymes occurs in a selective or nonselective manner. Chymotrypsin, trypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease, and en-dopeptidase Asp-N are frequently listed as selective enzymes, whereas thermolysin, pepsin, subtilisin, and elastase belong to the nonselective enzymes, although thermolysin preferentially cleaves peptide bonds before hydrophobic residues. 

Castberg, H. B. and Morris, H. A. 1976. Degradation of milk proteins by enzymes from lactic acid bacteria used in cheesemaking A review. Milchwissenschaft 31, 85-90. 

D18. Drozdz, R., and Naskalski, J. W., Inactivation and denaturation of some proteins by enzyme system Myeloperoxidase, chloride and hydrogen peroxide. Folia Histochem. Cytobiol. 31, 71-75 (1993). 

Fig. 23. Hypothetical scheme (20) for hydrolysis of proteins by enzymes. Here glycylglycine is shown chelated (55o) through the metal M to an enzyme molecule (heavy line). If the latter contains a negatively charged group close to the peptide bond, hydrogen ions may be accumulated at the latter and so accelerate hydrolysis of this link.

Bones are constantly subjected to forces that cause microscopic cracks. These microcracks (1) attach blood monocytes circulating within the periosteum and bone marrow and (2) induce adjacent osteoblasts to produce cytokines (Sect. 3.3.2) that cause these monocytes to proliferate, fuse, and differentiate into large multinucleated cells called osteoclasts. Osteoclasts cause bone resorption by acid demineralization and digestion of its proteins by enzymes that are optimally active in an acidic environment. These proteases and other hydrolytic enzymes are stored in a specialized, membrane-sealed compartment (lysosomes) into which they are guided by possessing terminal mannose 6-phosphate residues on N-linked glycans. 

C. Digestion of Proteins by Enzymes from Intestinal Cells... 

An ether-like combination of the amino acids was suggested by Nasse from the analogy between the hydrolysis of proteins by enzymes and that of the carbohydrates and fats. On account of the small number of hydroxyl groups in the molecules of the amino acids, which is limited to those contained in tyrosine, serine and oxyproline, such a combination can scarcely exist at any rate as the principal method of combination. 

These transformations of amino acid derivatives increases our interest in these compounds prepared by Curtius and his pupils, and gives an impulse to their further study, especially as formaldehyde is such an important compound in the synthesis of sugars by plants, and as the diamino acids and diamines occur as products of decomposition of proteins by enzymes and bacteria, although according to our present knowledge they are not formed in nature in this manner. 

Most reactions in cells are carried out by enzymes [1], In many instances the rates of enzyme-catalysed reactions are enhanced by a factor of a million. A significantly large fraction of all known enzymes are proteins which are made from twenty naturally occurring amino acids. The amino acids are linked by peptide bonds to fonn polypeptide chains. The primary sequence of a protein specifies the linear order in which the amino acids are linked. To carry out the catalytic activity the linear sequence has to fold to a well defined tliree-dimensional (3D) stmcture. In cells only a relatively small fraction of proteins require assistance from chaperones (helper proteins) [2]. Even in the complicated cellular environment most proteins fold spontaneously upon synthesis. The detennination of the 3D folded stmcture from the one-dimensional primary sequence is the most popular protein folding problem. 

Many globular proteins are enzymes They accelerate the rates of chemical reactions m biological systems but the kinds of reactions that take place are the fundamental reactions of organic chemistry One way m which enzymes accelerate these reactions is by bringing reactive func tions together m the presence of catalytically active functions of the protein... 

Peptides and proteins are formed by linking successive amino acids into chains or rings. The order (sequence) and types of amino acids (read from the sequence of codons) determine the chemical and physical properties of peptides and proteins (and enzymes). 

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

In contrast to the nicotinamide nucleotide dehydrogenases, the prosthetic groups FMN and FAD are firmly associated with the proteins, and the flavin groups are usually only separated from the apoen2yme (protein) by acid treatment in water. However, in several covalently bound flavoproteins, the enzyme and flavin coen2ymes are covalently affixed. In these cases, the flavin groups are isolated after the proteolytic digestion of the flavoproteins. 

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

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