Mechanism of Action of Bovine Testicular Hyaluronidase

The reactions of purified, homogeneous bovine testicular hyaluronidase have been studied with radioactively labeled oligomers of hyalobiuronic acid, (GlcUA-GlcNAc),, as substrates and acceptors. Transglycosylation occurs by transfer of a glycosyl residue with retention of configuration from a leaving group to an acceptor. On the basis of detailed examination of cleavage and transglycosylation patterns for the trimer; comparison of trimer, tetramer, and polymer as substrates; comparison of acceptors; equilibrium binding; and other data, it is proposed that the enzyme’s active site consists of five subsites for hyalobiuronate residues. In the terminology of Schechter, I., and Berger, A. ((1966) Biochemistry 5, 3371), these are s~-s,-s’~-s’~-s~, where the reducing terminus is to the right, and cleavage occurs between s, and s’,. It is proposed that subsite s’~ has a high affinity for a substrate residue, while s, and s’, have low substrate affinity, and sZ and s’, are intermediate in affinity. This proposal is seen to have mechanistic implications. The reactions of several substrates show similar bell-shaped pH dependences, with optima in the region of pH 5 to 5.5.