C-terminal residue, determining

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

The isoprenylation occurs at the Cys-residue of the consensus sequence Cys-A-A-X-COOH, whereby the nature of the C-terminal X-residue determines if farnesylation or geranylation occurs (fig. 3.13-1-14). After the isoprenoid residue is appended the three C-terminal residues are removed and the new COOH-group of the Cys-residue is methylated to increases the hydrophobicity of the C-terminus. A two-fold geranylation is found on two Cys residues of the Rab protein (see chapter 9). 

The acid-base behavior of peptides (10) is determined by the free a-amino group on the N-terminal residue, by the free a-carboxyl group on the C-terminal residue, and by the ionizable R groups located at intermediate positions. The pK values of the terminal a-carboxyl groups are somewhat higher and those of the a-amino groups somewhat lower than those of the corresponding free amino acids (Table 1) (11). 

In another successful case, Hexter and Westheimer (1971) were able to locate 5% of the total radioactivity in Tyr-146 after irradiation of [14C]diazoacetylchymotrypsin. The reaction is actually intermolecular, occurring in chymotrypsin dimers. Westheimer s group have determined the structure of several of the modified amino acids derived from the photolysis of proteolytic enzymes acylated with diazo reagents. Such data is not available for other photoaffinity reagents. Knowing that O-carboxy-methyl tyrosine was an expected insertion product Hexter and Westheimer (1971) were able to show that of the two Tyr residues in the chymotrypsin B chain only Tyr-146, the C-terminal residue, was modified. If the nature of the modified amino acid had not been known it would have been considerably more difficult to pin-point the site of photolabeling. 

All peptides and proteins, unless they are cyclic, contain the so-called N- and C-terminal residues a free a-amino group at one end and a free a-carboxyl group at the other. The identity of such groups may be determined by various chemical and enzymatic means. One of the first such methodologies utilized fluorodinitrobenzene (FDNB) for N-terminal group analysis ... 

A high-resolution structure has been determined for the BI IgG-binding domain of protein G. The structure comprises of four stranded /3-sheet made up of two antiparallel j8-hairpins connected by an a-helix. The two central strands of the sheet are parallel and comprise the N- and C-terminal residues. Comparison of the protein A and protein G IgG-binding domain architectures reveals no immediately obvious region that could take the place of the two interacting helices of protein A and protein G complex. 

The solution structure of the cytoplasmic domain of human ephrin-B2 was also determined (Song et al, 2002) and revealed that the 48 N-terminal ephrin residues are unstructured and are prone to aggregation. The highly conserved 33 C-terminal residues, on the other hand, form a well-packed hairpin structure followed by the flexible PDZ-binding tail. 

In order to determine the carboxy-terminus of Hez-PBAN,. 200 pmol of purified peptide was digested with carboxypeptidase P. Released amino acids were periodically analyzed as their PTC derivatives. Leu was found to be the C-terminal residue followed by Arg, Pro, Ser, Phe, Tyr, and Lys, respectively, thus confirming the automated Edman degradation data (Table I, run 5). However, none of the data could distinguish between a C-terminal amide or free acid. 

The most successful method of determining the C-terminal residue has been enzymatic rather than chemical. The C-terminal residue is removed selectively by the enzyme carboxypeptidase (obtained from the pancreaa), which cleaves only peptide linkages adjacent to free a/p/ra-carboxyl groups in polypeptide chains. The analysis can be repeated on the shortened peptide and the ney C-terminal residue identified, and so on. 

C-terminal racemisation of a peptide and specific deuteration of the C-terminal residue can be achieved by cyclisation of the peptide to the peptide oxazolone and quenching in 2H20. This specific reactivity of the C-terminal amino-acid residue has formed the basis of a C-terminal analysis of peptides the C-terminal residue is the only one to be racemised in this way and the identity of the C-terminal residue is revealed by analytical methods for determining d l ratios of amino-acid mixtures (Section 4.18.2 Sih and Gu, 1995). 

The amino acid sequence of a and /3 subunits of tropomyosin from rabbit skeletal muscle has been determined (Stone and Smillie, 1978 Sodek et al, 1978 Mak et al, 1980). Both subunits consist of a single peptide of 284 amino acid residues. The N-terminal residue is acetylated methionine, and the C-terminal residues is He. Assuming that all resi-... 

The C-terminal residue is determined by the use of either a chemical reagent or the enzyme carboxypeptidase. The... 

Carboxypeptidase is an exopeptidase that specifically hydrolyzes the C-terminal peptide bond and releases the C-terminal amino acid. Two problems are associated with its use the substrate specificity of the enzyme and the continuous action of the enzyme. The continuous action may yield the second, third, and additional residues from some chains even before the terminal residues on every chain are quantitatively released. Thus, it may be difficult to determine which residue is the C terminus. However, monitoring the sequential release of amino acids can often reveal the sequence of several residues at the C terminus. Concerning specificity, carboxypeptidase A releases all C-terminal residues except Lys, Arg, and Pro carboxypeptidase B cleaves C-terminal Arg and Lys residues and carboxypeptidase C hydrolyzes C-terminal Pro residues. Thus, more than one method may be needed to establish the C-terminal amino acid. 

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