STUDIES ON THE SPECIFICITY OF ACETYLAMINOACYL-PEPTIDE HYDROLASE

In a continuing attempt to explore the types of specificity determinan ts that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substr ates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). T he reference substrate was Ac-AAAA, and most of the other substrates w ere derived from this basic structure by single amino acid substitutio ns. The K-m and k(cat) for the different substrates were determined by standard steady-state kinetics, and the corresponding Delta Delta G(T ) double dagger value derived from k(cat)/K-m was used for the compari son, setting Delta Delta G(T) double dagger for Ac-AAAA equal to 0. Th e best substrates were found to be those containing negative charges ( Asp > Glu) or aromatic residues in positions 1', 2', or 3' (Delta Delt a G(T) double dagger values of 2-5 kJ); the negative charge provided b y 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 G( T) double dagger values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inh ibitory effect of positive charges in similar peptides (Krishna RG, Wo ld F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interact ions. In assessing all the data, it is clear that the properties of th e individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position. Thus, whi le the peptides with single Asp or Phe substitutions in 1', 2', and 3' gave Delta Delta G(T) double dagger values of 3-5 kJ, the peptide con taining all 3 modifications, Ac-ADDF, gave only 1 kJ. Similarly, Ac-TA AA was a poor substrate and Ac-GAAA was not cleaved at all in this stu dy, while in the past other peptides such as Ac-TGG and Ac-GGG have be en found to be excellent and reasonably good substrates, respectively. Although the rate differences observed in this work are minor, they n evertheless appear to reflect the kind of structural detail that is in volved in determining the specificity of protein-protein (peptide) int eractions.