AbstractIn a continuing attempt to explore the types of specificity determinants that may affect protein‐protein (peptide) interactions, a number of short (2‐5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl‐peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac‐AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat, for the different substrates were determined by standard steady‐state kinetics, and the corresponding δδGT D̊x value derived from kcat/Km was used for the comparison, setting δδGT D̊ for Ac‐AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1′, 2′, or 3′ (δδGT D̊ values of 2‐5 kJ); the negative charge provided by the C‐terminus of the substrate also appears to be important, since the amide and O‐Me ester derivatives caused a change in δδGT D̊ values of ‐7 to ‐8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582‐589), and the proposed active site model incorporates subsites for both charge‐charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make‐up of each peptide and not only the effect of a single residue in a given position. Thus, while the peptides with single Asp or Phe substitutions in 1′, 2′, and 3′ gave δδGT D̊ values of 3‐5 kJ, the peptide containing all 3 modifications, Ac‐ADDF, gave only 1 kJ. Similarly, Ac‐TAAA was a poor substrate and Ac‐GAAA was not cleaved at all in this study, while in the past other peptides such as Ac‐TGG and Ac‐GGG have been found to be excellent and reasonably good substrates, respectively. Although the rate differences observed in this work are minor, they nevertheless appear to reflect the kind of structural detail that is involved in determining the specificity of protein‐protein (peptide) interactions.