Glutamic acid cleavage

The binding site of lysozyme will accommodate six hexose residues (A-F). The carboxyl groups of glutamic acid-35 and aspartic-52 are located between sites D and E. The lactyl groups at C-3 of the NAM residues cannot fit in sites A, C, and E. Therefore NAM residues must be located at sites B and D. Cleavage occurs at the reducing end of the NAM residue in site D (the bond being broken is between residues D and E). 

The binding of glycyl-L-tyrosine in the active site pocket of carboxypeptidase A is illustrated in Fig. 15. Tyrosine-248 and glutamic acid-270 are believed to participate in the catalytic reaction and represent the acidic and basic groups, respectively, involved in the bell-shaped pK-rate profile. In the bond-cleavage reaction, the carboxyl group of Glu-270 may act by a nucleophilic attack on the carbonyl group while Tyr-248... 

The introduction of a glutamic acid (E bold), after each RADI 6 repeat was made to allow cleavage by endoproteinase Glu-C, which cleaves C-terminal to glutamate. A cellulose binding domain (CBD) (Table 1) was selected as the affinity tag, as CBDs bind strongly and specifically to cellulose which is a relatively cheap and abundant purification matrix. The main problem encountered was the low level of peptide recovered. Theoretically, 1 g of fusion protein should give 267mg of peptide, but only 10.1 mg of peptide was recovered after RP-HPLC. 

All data obtained with Tecan Ultra Evolution MTP reader. The following excitation and emission wavelengths were used EDANS and AMC 350 and 500 nm RhllO 485 and 535 nm TAMRA 535 and 595 nm PT14 405 and 450 nm. 4 = primary cleavage site confirmed by MS. AMC = aminomethylcoumarin. RhllO = rhodamine 110. yE = glutamic acid attached to RhllO via its carbonic acid in side chain. EDANS = fluorophore 5-[(2-aminoethyl)amino]naphthalene-l-sulphonic acid. DABCYL = 4-(4-dimethylaminophenylazo)benzoic acid quencher. BTN = biotin. PT14 = acridone-based fluorescence lifetime label. 

TOF-TOF instruments can also analyse small intact proteins but this instrument leads to low sequence coverage. Indeed, as observed for CID, proteins above 5000 Da produce sequential b and y fragment ions principally at the termini of the protein. Nevertheless, the most predominant fragment ions correspond to cleavage at the C-terminal to aspartic or glutamic acid residues and at the N-terminal to proline residues in the protein [73],... 

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