Enzymes partial proteolysis

In mammalian cells, the two most common forms of covalent modification are partial proteolysis and ph osphorylation. Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, proteolysis constitutes an irreversible modification. By contrast, phosphorylation is a reversible modification process. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases. 

Table 9.1 lists some of the other enzymes activated by partial proteolysis. A common pattern is for proteolysis to occur in a loop that connects two different domains of the protein, relieving a constraint that interferes with the formation of the active site. 

The enzymes that participate in blood clotting also are activated by partial proteolysis, which again serves to keep them in check until they are needed. The blood coagulation system involves a cascade of at least seven serine proteases, each of which activates the subsequent enzyme in the series (fig. 9.2). Because each molecule of activated enzyme can, in turn, activate many molecules of the next enzyme, initiation of the process by factors that are exposed in damaged tissue leads explosively to the conversion of prothrombin to thrombin, the final serine protease in the series. Thrombin then cuts another protein, fibrin, into peptides that stick together to form a clot. 

Partial proteolysis, an irreversible process, is used to activate proteases and other digestive enzymes after their secretion and to switch on enzymes that cause blood coagulation. Common types of reversible covalent modification include phosphorylation, adenylyla-tion, and disulfide reduction. 

Based on the work done in our laboratory, we believe that multiple enzymes of the same type are derived from the same enzyme and potentially arise from partial proteolysis of such an enzyme (10). In previous studies, we have purified three distinct cellobiases from T. reesei which are chromatographically distinct yet kinetically similar. 

By) (24) and partial proteolysis by exposure to trypsin or chymo-trypsin destroys the combined activity to a greater extent than the glucosidase activity (38). These results also suggest that the transferase and the glucosidase activities are located at separate sites on the same enzyme molecule. 

Partial proteolysis has been used by several researchers to improve functional properties, i.e. foaming, solubility of proteins (7,8,9). The significant problems associated with enzyme hydrolysis of proteins are excessive hydrolysis occurring under batch conditions, the generation of bitter flavors during hydrolysis and the cost of enzymes. Extensive information on factors affecting proteolysis of proteins and the problem of bitterness has been reviewed by Fujimaki et al. (7) in conjunction with studies of the plastein reaction. 

Enzyme hydrolysis is occasionally used to modify the functional properties of proteins and yeast autolyzates are used commercially as food flavorants (66,86). Partial proteolysis of... 

In addition to the problem of partial proteolysis, the solubility of larger unprotected peptide segments may be crucial in the enzyme-assisted approaches, since solubilization with... 

Partial proteolysis of soybean proteins with endopeptidases has been used to remove flavor compounds and related fatty materials from soybean curd and defatted soybean flour (21). Certain soybean protein concentrates possess an undesirable beany and oxidized flavor. Treatment of soybean curd and defatted soybean flour with endopeptidases such as aspergillopeptidase A released off-flavor compounds such as 1-hexanal and 1-hexanol which could be removed from the hydrolysate by solvent extraction. The enzymically digested products had less odor, taste, and color than the starting material and were more stable to oxidative deterioration. 

Partial proteolysis of soy protein isolate with neutral protease from Aspergillus oryzae altered certain functional properties ( ). Solubility was increased in the enzyme-treated soy isolate at both neutral pH and at the isoelectric point (pH... 

Partial reactions for the yeast Type I synthetase were studied in a classic series of experiments by Lynen (1967). Peptides capable of partial reactions have been isolated from the yeast and chicken liver enzymes (cf. Wakil et ai, 1983). Particular interest has been focused on the thioesterase. This enzyme, more easily isolated by partial proteolysis than those for most of the other partial reactions, is important in at least partly regulating chain length termination. Thioesterases isolated from several animal fatty acid synthetases have very similar amino acid sequences around the active serine residue (Poulose et al, 1981). The medium-chain fatty acids produced by synthetases from some mammary glands appear to be due to thioesterase II (a second thioesterase) (Libertini and Smith, 1978). 

Fig. 2. Partial proteolysis of C. botulinum enzyme substrates with Staphylococcus V8 protease. A portion of PC12 lysate was labeled as described in Fig. 1 and fractionated on a 10% SDS-PAGE tube gel. The tube was placed on top of a 15% SDS-PAGE slab gel and overlaid with 100 p-g/ml Staphylococcus V8 protease (14) before fractionation in the second dimension.

The enzyme that causes the partial proteolysis of procollagen and removes the disposable 20% of residues may be present extracellularly, but so far it has not been identified. 

The partially-purified extract oxidised a range of phenolic substrates, and also contained proteinases and amino acid decarboxylases. Preincubation of a toluene-treated soil enzyme preparation for 12h at 37°C did not affect diphenol oxidase activities, ie. the oxidases appeared to be resistant to attack by the coextracted soil proteinases. Addition of hyaluronidase before preincubation also was without effect. Preincubation with the microbial proteinase, Pronase for I8h at 37°C decreased diphenol oxidase activities by 307o, and by 100% when both Pronase and hyaluronidase were added. The results suggested that the polysaccharides associated with the extracted soil oxidases protected the enzymes from proteolysis and may play a role in stabilizing exocellular enzymes in soils. 

A protein known as the amyloid precursor protein (APP) spans the plasma membrane of the neurone. It possesses an extracellular domain but its function is unknown. The extracellular domain is partially hydrolised by proteolytic enzymes, known as secretases. One of the products is the amyloid peptide, of which there are two forms. The larger form, contains 42 amino acids and readily polymerises to form plaques in the extracellular space, damaging the neurones. Some sufferers possess a mutated form of the APP protein which more readily produces the larger peptide upon proteolysis, so that more toxic plaques are produced. It is the progressive accumulation of these plaques that is considered to be one cause of Alzheimer s disease. 

Evidence demonstrating that the degradation observed is catalyzed by enzyme(s) was obtained by typical denaturing treatments. Late extracellular protein fractions from both strains present the same characteristics resistance to heat up to 100°C, partial resistance to acidity as low as pH 1.0 (samples being returned to pH 7.8 prior to assaying for activity), but are completely inactivated by proteolysis with a mixture of trypsin and chy-motrypsin (Table II). 

Frequently a third enzymatic digestion is necessary to eliminate all the possible ambiguities. Using trypsin as a proteolysis enzyme allows one partially to distinguish Lys from Gin, two isobars. In fact, the trypsin specifically cleaves on the C-terminal side of Lys and Arg, so all the amino acids with mass 128 ending a peptide can be identified as Lys. However, the absence of a cleavage cannot be used as proof in identifying an amino acid... 

Loss of translocation competence could result from burial of the signal peptide within the folded precursor, but at least in the case of preMBP, the signal peptide is still accessible as determined by its selective sensitivity to proteolysis and its ability to bind amphiphiles (Dierstein and Wickner, 1985). In the case of the methotrexate-stabilized COX/DHFR fusion, the COX target peptide is clearly accessible since the precursor binds to energized mitochondria (a target peptide-dependent reaction), and the target peptide is susceptible to proteolysis by partially purified matrix processing enzyme (Eilers and Schatz, 1986). 

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