Amino acids, isolation groups, determination

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

Studies of the structure of papain were initiated by Thompson (165), who determined the amino or N-terminal amino acid sequence and the free amino groups. Crystalline papain and mercuripapain were allowed to react with l-fluoro-2,4-dinitrobenzene (FDNB) by the procedures of Sanger (136). The dinitrophenyl (DNP) proteins were subsequently hydrolyzed in 6 iV HCl and the DNP-amino acids isolated by partition chromatography of the ether extracts of the hydrolyzates. Quantitative estimates of the DNP-amino acids were made spectrophotometrically. The results are given in Table V. 

In subsequent studies (Kenney et ai, 1974a, 1976a) of the flavin prostjietic group of the j -cyclopiazonate oxidocyclases, the isoenzymes were digested with trypsin and chymotrypsin. A flavin mononucleotide peptide was isolated from the resultant mixture by chromatography on Florisil and diethylaminoethyl cellulose and by hydrolysis with nucleotide pyrophosphatase. Its amino acid composition was determined after hydrolysis in... 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

Sequence Determination of the Brain Peptide Leucine Enkephalin A group of peptides that influence nerve transmission in certain parts of the brain has been isolated from normal brain tissue. These peptides are known as opioids, because they bind to specific receptors that also bind opiate drugs, such as morphine and naloxone. Opioids thus mimic some of the properties of opiates. Some researchers consider these peptides to be the brain s own pain killers. Using the information below, determine the amino acid sequence of the opioid leucine enkephalin. Explain how your structure is consistent with each piece of information. 

Chemical methods for carboxyl end-group determination are considerably less satisfactory. Treatment of the peptide with anhydrous hydrazine at 100°C results in conversion of all the amino acid residues to amino acid hydrazides except for the carboxyl-terminal residue, which remains as the free amino acid and can be isolated and identified chro-matographically. Alternatively, the polypeptide can be subjected to limited breakdown (proteolysis) with the enzyme carboxypeptidase. This results in release of the carboxyl-terminal amino acid as the major free amino acid reaction product. The amino acid type can then be identified chroma-tographically. 

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