Tryptic hydrolysis residues

Pathy, L., and Smith, E.L. (1975) Reversible modification of arginine residues Application to sequence studies by restriction of tryptic hydrolysis to lysine residues./. Biol. Chem. 250, 557. 

Glycine A-methyltransferase is also reported to have an ordered binding mechanism with SAM binding first to the enzyme, there being no metal-ion dependency. Cooperative behavior is observed with SAM binding. The cooperative nature can be eliminated by the tryptic hydrolysis of the N-terminal eight amino acid residues. 

The primary structure of the t chain is not established, and only results of preliminary structural analyses have been reported (H30, K15). Extensive studies of the chain have revealed rather unique structural features (C2). Its amino acid composition, for instance, differs from those of all other hemoglobin chains and is, among others, characterized by the presence of five isoleucyl residues. Tryptic hydrolysis produced several peptides with amino acid compositions not observed for any of the tryptic peptides of the a, y, or 8 chain, although other peptides had compositions identical to those of tryptic peptides of the y chain. There seems to be no doubt that the structures of the e and chains are different (C2). 

The rate of hydrolysis is affected by the amino acid that follows the basic residue in the sequence. Thus the Arg-Asp bond is cleaved more slowly than most other peptide bonds on the right side of arginine. Similarly, the bond between arginine and cysteic acid (the oxidized form of cysteine) is relatively resistant to tryptic hydrolysis. As it will be shown later such differences can be exploited in sequence studies, but it is possible to create even more substantial differences by appropriate modifications of the peptide molecule. For instance, treatment of a peptide that contains both lysine and arginine residues with maleic acid anhydride or preferably with citraconic acid anhydride yields derivatives in which the lysine residues have no free amino group in their side chain... 

Tryptic hydrolysis can hardly be expected to give accurate information on the number of consecutive arginine residues in a protamine molecule. Thus, a complementary method of partial hydrolysis had to be developed so as to obtain longer peptide fragments in which intact sequences of arginine are retained. 

Fig. 12. Tryptic map of it-PA (mol wt = 66,000) showing peptides formed from hydrolysis of reduced, alkylated rt-PA. Separation by reversed-phase octadecyl (C g) column using aqueous acetonitrile with an added acidic agent to the mobile phase. Arrows show the difference between A, normal, and B, mutant rt-PA where the glutamic acid residue, D, has replaced the normal arginine residue, C, at position 275.

The arrangement of S965, H746, and the oxyanion hole suggests that the classical steps of peptide-bond hydrolysis follow the sequence of the trypsin-like serine proteases, namely the formation of the tetrahedral adduct, the acyl-enzyme complex, and hydrolysis. Tricorn has been shown to exhibit both tryptic and chymotryp-tic specificities (Tamura et al. 1996a). The X-ray structure reveals that specificity for basic PI residues is conferred by D936 which is provided by the diad-related subunit (see Figures 10.9 and 10.10). 

Fig. 3. Limited and extensive peptic hydrolysis of two tryptic peptides from human hemoglobin )8-chains (Goldstein et al., 1963). Peptide A contains residues 105-120 and peptide B contains residues 83-104 of the j8-chain.

Arginine residues modified by nitromalondialdehyde are resistant to tryptic digestion. However, reduction of the derivative by sodium borohydride yields tetrahydropyrimidyl compounds which are susceptible to hydrolysis by trypsin. Modification by nitromalondialdehyde thereby permits the restriction of tryptic digestion to lysine... 

---