Specificity and mode of action of the muscle-type protein-arginine deiminase.

The primary and secondary specificities and mode of action of the muscle-type protein-arginine deiminase (PAD) were investigated using various derivatives of Arg and its homologues, as well as Arg-containing peptides by quantitative analyses of the reaction products on reverse-phase HPLC. The enzyme converted benzoyl-D-Arg-p-nitroanilide into its citrulline derivative at 18% of the rate of the L-isomer, while the D-Arg residues in peptides were not deiminated to a significant extent. This suggests that PAD does not have strict stereospecificity and it is dependent on the structure of the residues or groups on both sides of the target Arg residue. In contrast, the benzoyl-/-ethyl ester derivatives of homoarginine, alpha-amino-beta-guanidino-propionic acid, canavanine, and NG-methyl-Arg, exhibited poor PAD susceptibility, suggesting that the length and nature of the arm as exactly three CH2 groups, and the integrity of the guanidyl group are quite strict specificity determinants. The enzyme action on Arg residues in peptides depends greatly on their position in the sequence, and on the nature of the neighboring residues. For example, deimination of Arg residues situated at positions 1-3 from the NH2-terminus, except for those preceded by a carbobenzoxy- or benzoyl-group, were in most cases very slow, whereas those at the COOH-terminus were deiminated relatively faster. A single Arg residue sandwiched between two Pro residues was not deiminated at all, while a pair of Arg residues between two Pro were deiminated moderately. Consequently, PAD exhibited a variety of modes of action on more than one Arg residues in the peptides tested. The results suggest the applicability of PAD, albeit quite limited, for selective modification of certain Arg residues in peptides and proteins by appropriately controlling reaction time and several other parameters. The PAD's mode of action was compared with those of three Arg-bond cleaving proteases.