Studies on the specificity of acetylaminoacylpeptide hydrolase

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Abstract
In 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 delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ 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' (delta delta GT++ 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 delta delta GT++ 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Funding Information
  • Robert A. Welch Foundation (AU-916, AU-0009)
  • USPHS (GM-31305)