A chimeric alpha-glucan phosphorylase of plant type L and H isozymes. Functional role of 78-residue insertion in type L isozyme.

Higher plant tissues such as potato tuber and leaf contain two alpha-glucan phosphorylase isozymes designated types L and H. Although the sequences of the two isozymes are highly conserved except for a 78-residue insertion found uniquely in the type L isozyme, they differ strikingly in affinities for substrates. To examine whether the insertion in the type L isozyme plays a role in enzymic functions, particularly in substrate specificities, we have constructed a chimeric enzyme, in which a 189-residue sequence of the type L isozyme including the insertion and its flanking regions is replaced by the corresponding sequence (112 residues) of the type H isozyme lacking the insertion. The gene for the chimeric enzyme as well as the cDNA for the type L isozyme were expressed at a low temperature in Escherichia coli cells under the control of the strong T7 RNA polymerase promoter. The purified chimeric phosphorylase was five times less active than the parent type L isozyme, but its affinity for glycogen was much higher than that of the type L isozyme and only slightly lower than that of the type H isozyme. The Michaelis constants of the chimeric enzyme for small oligosaccharides were comparable with those of the type L isozyme. These results provide evidence for the role of the 78-residue insertion in the type L isozyme, lowering the affinity of the enzyme for large, branched substrates probably through steric hindrance. It is also assumed that the corresponding region in the type H isozyme contains a high affinity site like the glycogen storage site occurring in the animal enzyme.