Enzymatic cleavage, protein structure

Another application area in microwave technology is the use of microwave irradiation for the enzymatic digestion of proteins [106]. As discussed in the early part of this chapter, enzymatic cleavage to produce smaller peptide fragments of protein samples is an important step in structural characterization of proteins [24]. Traditional enzymatic digestion method usually takes several hours, whereas microwave-assisted digestion occurs in minutes. The initial... 

The peptides obtained by specific chemical or enzymatic cleavage are separated by some type of chromatography. The sequence of each purified peptide is then determined by the Edman method. At this point, the amino acid sequences of segments of the protein are known, but the order of these segments is not yet defined. How can we order the peptides to obtain the primary structure of the original protein The necessary additional information is obtained from overlap... 

HIV-1 PR cleaves the multidomain protein encoded by the virus genome to yield separated structural proteins. Structure-based drug-design studies have shown that in the substrate-cleavage site - two Asp-Thr-Gly loops at the subunit-subunit interface (Fig. 5a) - the almost coplanar conformation of the catalytic Asp dyad is crucial for enzymatic function and for the binding of both substrate and inhibitors [88-90]. 

The sequence of amino acids in the polypeptide chain (i.e., the primary structure of a polypeptide or protein) can be established by selective chemical and enzymatic cleavage of the protein followed by separation, amino acid analysis, and sequence determination of all peptide fragments. The entire amino acid sequence is established by overlapping identical regions of the individual fragments. The following problem illustrates the procedure. 

It is not always possible to apply enzymatic hydrolysis directly to proteins as they are in the native form. Native, globular proteins (e.g., from soy, corn, almond) or fibrous insoluble proteins (e.g., collagen, keratins, elastin) are generally resistant to proteolysis this is generally explained by the compact tertiary structure of the protein that protects most of the peptide bonds. In the denatured, unfolded form the peptide bonds are exposed and available for enzymatic cleavage. As native proteins in aqueous solution are in dynamic equilibrium with a number of more or less distorted forms, part of which can be considered denatured and thereby accessible to enzyme attack, the initial break of a few peptide bonds can destabilize the protein molecule and cause irreversible unfolding in some cases (e.g., hydrolysis of egg albumin by pepsin) this mechanism allows the protease to perform the hydrolysis to a remarkable extent. More frequently, especially when covalent bonds (disulfide bonds) stabilize the native form of the protein, a preliminary partial or extended denaturation is needed to make enzymatic hydrolysis possible this is normally achieved by heating or chemical attack, or a combination of the two. 

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