Enzyme residue determination

In addition to a-l-PI, there are other examples of the presence of Met(O) residues in proteins isolated from biological material. Proteins found in lens tissue are particularly susceptible to photooxidation and because of the long half-lives of these proteins, any oxidation could be especially detrimental. In this tissue, protein synthesis is localized to the outer region of the tissue and most proteins are stable for the life of the tissue - ". It is thus somewhat surprising that not only is there no Met(O) residues in the young normal human lens but even in the old normal human lens only a small amount of Met(O) residues is found . However, in the cataractous lens as much as 65% of the Met residues of the lens proteins are found in the form of Met(0) . Whether this increase in Met(O) content in these proteins is a cause or a result of the cataracts is not known. In order to determine whether the high content of Met(O) in the cataractous lens is related to a decreased activity of Met(0)-peptide reductase, the level of this enzyme was determined in normal and cataractous lenses. It can be seen from Table 9 that there are no significant differences between the levels of Met(0)-peptide reductase in normal and cataractous lenses. In spite of these results, however, it is still possible that the Met(0)-peptide... 

The enzyme has been well characterized, the complete amino acid sequence is known, and there have been many crystal structure determinations. The complete amino add sequence for the liver enzyme was determined by Jornvall in 1970 1335 it contained 374 residues, consistent in... 

The NH2-terminal residue of the enzyme was determined by the cyanate-hydantoin procedure of Stark and Smyth (40) and by Edman... 

The COOH-terminal residue, determined both with carboxypeptidases A and B (43), was lysine (5.4 moles/mole of enzyme), and the penultimate residue was alanine. Additional components were not detected in either digestion (34). 

Enzymes are made from just 20 a-amino acid building blocks (structures and abbreviations are shown in Table 5.1). Each amino acid has a unique side chain, or residue, which can be polar, aliphatic, aromatic, acidic, or basic. The amide bonds (peptide bonds) make up the enzyme s backbone, and the residues determine the ultimate structure and catalytic activity of the enzyme. When the sequence of amino acids (the primary structure) for an enzyme is assembled in vivo, it folds... 

The substrate specificity of an enzyme is determined by the properties and spatial arrangement of the amino acid residues forming the active site. The serine proteases trypsin, chymotrypsin and elastase cleave peptide bonds in protein substrates on the carboxyl side of positively charged, aromatic and small side-chain amino acid residues, respectively, due to complementary residues in their active sites. 

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase (see Topic C5). These three enzymes belong to a family of enzymes called the serine proteases - serine because they have a serine residue in the active site that is critically involved in catalysis and proteases because they catalyze the hydrolysis of peptide bonds in proteins. The three enzymes cleave peptide bonds in protein substrates on the carboxyl side of certain amino acid residues. 

Two recently isolated serine proteases have quite different specificities. One is a protease of Staphylococcus aureus which has a high specificity for glutamic acid residues at Pi 16, 17). The other is the yeast carboxypeptidase listed in Table I 11), As the name indicates, it degrades polypeptides by cleaving amino acid residues from the C-terminal end of the chain—a most unexpected specificity for a serine protease. Unlike Carboxypeptidase A, it is stable, is capable of removing proline residues, and would seem to be an ideal enzyme for determining C-terminal sequences. 

Figure 1 Diagram of a protease active site. A protease cieaves a peptide at the scissiie bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminai side of the scissiie bond and their prime-side residues C-terminal to the scissiie bond. The cataiytic residues determine the ciass of protease. Serine, cysteine, and threonine proteases hydroiyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, giutamic and metaiioproteases activate a water moiecuie to hydroiyze the peptide bond in a non-covalent manner.

Although chemical techniques have provided a considerable amount of dais on the structure of galactomannans, it has required the use of highly purified enzymes to determine the distribution of the side chains. Studies using a-D-galactosidases have indicated that the galactomannans from Gleditsia ferox and locust bean have a different distribution of a-D-Ga p residues (Courtois and Le Dizet, 1966). 

Thermal stability of soluble or immobilized enzyme was determined by incubating the biocatalyst in 100 mM sodium phosphate buffer pH 7.0 at 50 or 60 °C. Periodically, samples were withdrawn, and their residual activities were assayed by the hydrolysis of methyl butyrate. Residual activity is given as percentage of initial activity (hydrolytic activity before incubation). Thermal deactivation curves have been described following the... 

In summary, to date there is neither a plausible concept of how different mutations in the same gene cause such disparate clinical symptomatology of the disease on one hand (lethal-neonatal versus adult-onset) nor how such different residual activities of this enzyme lead to comparably similar phenotypes (adult-onset muscular) on the other hand. A variable degree of reduction of CPT activity, variable posttranslational modifications of the enzyme in different tissues, a disturbance of regulatory properties of the CPT system or a variable efHeiency of further distal eomponents of the P-oxidationmachin-ery by an pathologieally altered enzyme might determine the difference in clinical severity of the different forms of CPT II deficiency. 

Before the structure of the enzyme was determined, chemical modification experiments (using proteolysis and mass spectrometry to identify the modified residue) and site-directed mutagenesis were used to identify Arg 21, Tyr 28, and a His as catalytically important side chains.Substrate and solvent H isotope effects, proton inventory studies, and the pH-dependence of the kinetic parameters and were used to identify the enzyme mechanismas an E]CB elimination reaction proceeding via an enolate intermediate, with the initial proton abstraction as the rate-limiting step. The unusual sharp increase in and above pH 9 was interpreted as the effect of an active-site Arg side chain on the basicity of a His. " ... 

Domain Residue DNA Binding Residues Auto- NAD modification Binding Residues Residues Native Enzyme Calculated Determined Residues (%) (%) ... 

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