Limited proteolysis of S-adenosylhomocysteine hydrolase: implications for the three-dimensional structure.
S-Adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited proteolytic digestion utilizing the proteases trypsin, V8, and papain. Results of the trypsin digest revealed one major susceptible peptide bond between amino acid residues 103 and 104 which is most likely exposed to solvent. Binding of the substrate adenosine substantially reduced the susceptibility of this site, indicating that this peptide bond may be located at or near the substrate binding site. Wild-type AdoHcy hydrolase (which exists as a tetramer) was completely resistant to V8 digestion, while a site-directed mutant form (in which Lys at position 426 was changed to Glu) of the enzyme that exists primarily as a monomer had one major V8 protease cleavage site between amino acid residues 198 and 199, suggesting that these two amino acid residues may be positioned within the tetramerization region of each subunit. Limited papain digestion of AdoHcy hydrolase revealed that the enzyme, despite multiple peptide bond cleavages, was able to maintain its quaternary structure and remain catalytically functional. This observation suggests that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding. Identification of a surface-exposed peptide bond and one located in the tetramerization domain of each subunit may provide some constraints on how each subunit can be oriented in space. Results from this study support a previously described model (D. B. Ault-Riché, C. S. Yuan, and R. T. Borchardt (1994) J. Biol. Chem., 269, 31, 472-31, 478) in which the formation of the active site is dependent upon proper quaternary structure and also suggest that the active site of the enzyme may be located at or near the tetramerization domain of each subunit.