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.
Mori
H
H
Institute of Scientific and Industrial Research, Osaka University, Japan.
Tanizawa
K
K
Fukui
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D006005:Phosphorylases / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014675:Vegetables / Q000201:enzymology*
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D004247:DNA; D006005:Phosphorylases