AbstractIn an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl‐peptide hydrolase (EC 3.4.19.1), a number of acetylated peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were d‐Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of peptides, by extension of the N‐terminus with acetylamino acids or acetylpeptides, activated as N‐hydroxysuccinimide esters, or by isolation of the N‐terminal peptides from naturally occurring acetylated proteins. It was found that d‐Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated peptides with d‐Ala in positions 3 or 4 were as good substrates as those containing l‐Ala. Peptides with Pro in positions 2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac‐AAP gave reasonably high activity (30% of Ac‐AAA), which was reduced to 1–2% if additional residues were present at the C‐terminus (Ac‐AAPA, Ac‐AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in hydrolase activity varying with the charge's distance from the N‐terminus from 0 to 15–20% of the rates obtained with the reference peptides without positive charges. Deprotonation of His at high pH generated excellent substrates, and removal of the positive charges of Lys by acetylation or, even better, succinylation also gave improved substrate quality, demonstrating that the positive charges are responsible for the inhibition. Long peptides (10–29 residues) were generally found to be poor substrates, especially when they contained positive charges and Pro. The better long peptide substrates do not have these residues, but contain negative charges instead. A survey of the N‐terminal sequences of more than 100 acetylated proteins revealed that about 95% of them have Pro and/or positively charged amino acids among the first 10 residues, suggesting that these residues may be natural inhibitors of hydrolase action in vivo. In addition to the specific and large effect of the residues described above on substrate quality, it also appears that there is a general effect of the overall sequence of each peptide, and that the specific effects of individual residues are modulated significantly by the environment (context) in which they are expressed.