Specificity determinants of acylaminoacyl‐peptide hydrolase

Abstract

In 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. l), 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 Nhydroxysuccinimide 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 1-2070 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 d o 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.Keywords: acetylated N-termini; acylaminoacyl-peptide hydrolase; N-terminal processing; posttranslational modification; primary sequence specificity determinants A large number of chemical modifications of proteins transport, and during proteolytic destruction. Most of take place in living cells both during co-and posttransthese reactions are characterized by an extr...

Studies on the specificity of acetylaminoacylpeptide hydrolase

“…This conclusion is consistent with the data mentioned above (Krishna & Wold, 1992) showing that positively charged residues (Arg or His) are strongly inhibitory in positions Pl', P2', and P3' and even in positions further away from the scissile bond. The effect of pH on the inhibition by His showed unequivocally that it is the positive charge and not the bulky imidazole ring that causes the inhibition.…”

“…Values calculated from the data of Krishna and Wold (1992). &Wilson, 1972;Tsunasawa et al, 1975;Gade & Brown, 1978;Kobayashi & Smith, 1987;Radhakrishna & Wold, 1989).…”

“…In this connection it is of interest to reconsider in a little more detail the His effects that were discussed above. Performing the standard assay with His-containing peptides between pH 5.0 and 8.7 demonstrated that the positively charged side chain (Ac-AH'A and Ac-AAH+A) supported no activity, whereas the neutralized His-containing peptides (Ac-AHA and Ac-AAHA) containing the aromatic side chain in fact showed considerably more activity than did the Ac-AAA or Ac-AAAA controls (Krishna & Wold, 1992). In designing the active site for the enzyme, it thus appears that both an ionic interaction subsite and a hydrophobic interaction subsite need to be considered.…”

“…The specificity of the hydrolase is first of all determined by the acetylated amino acid to be hydrolyzed, showing strong preference for the most commonly occurring acetylated N-termini Krishna & Wold, 1992). However, the residues C-terminal to the scissile bond also contribute significantly to the specificity (Gade & Brown, 1978;Schonberger & Tschesche, 1981;Kobayashi &Smith, 1987;Radhakrishna & Wold, 1989;Krishna & Wold, 1992).…”

“…However, the residues C-terminal to the scissile bond also contribute significantly to the specificity (Gade & Brown, 1978;Schonberger & Tschesche, 1981;Kobayashi &Smith, 1987;Radhakrishna & Wold, 1989;Krishna & Wold, 1992). These contributions can be summarized as follows.…”