LIMITED PROTEOLYSIS OF S-ADENOSYLHOMOCYSTEINE HYDROLASE - IMPLICATIONS FOR THE 3-DIMENSIONAL STRUCTURE

S-Adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited pro teolytic digestion utilizing the proteases trypsin, V8, and papain. Re sults of the trypsin digest revealed one major susceptible peptide bon d 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 hy drolase (which exists as a tetramer) was completely resistant to V8 di gestion, while a site-directed mutant form (in which Lys at position 4 26 was changed to Glu) of the enzyme that exists primarily as a monome r had one major V8 protease cleavage site between amino acid residues 198 and 199, suggesting that these two amino acid residues may be posi tioned within the tetramerization region of each subunit. Limited papa in digestion of AdoHcy hydrolase revealed that the enzyme, despite mul tiple peptide bond cleavages, was able to maintain its quaternary stru cture and remain catalytically functional. This observation suggests t hat AdoHcy hydrolase exists as a very compact enzyme with extensive in tramolecular bonding, Identification of a surface-exposed peptide bond and one located in the tetramerization domain of each subunit may pro vide some constraints on how each subunit can be oriented in space, Re sults from this study support a previously described model (D. B. Ault -Riche, 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 up on proper quaternary structure and also suggest that the active site o f the enzyme may be located at or near the tetramerization domain of e ach subunit. (C) 1995 Academic Press, Inc.