Type-H Isozyme

The DNA sequence around the initiation ATG codon of the type-H isozyme cDNA is rather dissimilar to that of the type-L isozyme cDNA and the consensus sequence for plant genes proposed by Heidecker and Messing.69 The difference in the sequences of this region may be correlated with the difference in the expression levels of each isozyme in potato tuber. 

Sequence analysis of the 5 -upstream regions of the genomic DNAs of the type-H and -L isozymes (unpublished results) indicated that in the type-L isozyme gene there is a significantly homologous region with the 5 -upstream region of class II patatin,81 which is 

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Amino acid sequence comparison of a-glucan phosphorylases. H, the potato type-H isozyme L, the potato type-L isozyme and R, rabbit muscle phosphotylase.791 The type-H isozyme sequence is used as the reference sequence only the amino acid residues that are nonidentical in the type-L isozyme and rabbit muscle enzyme are indicated. (From Biol. Chem., 266 (28), 18453 (1991)). 

Fig. 6.2 Spatial structure of the active site of rabbit muscle phosphorylase85 (A) and sequence comparison of the active site residues (B). A, Hydrogen bonds of less than 3.3 A (dotted lines) and water molecules (crosses) are also indicated. B, Sequences constituting the active-site region are compared among potato type-H isozyme (H), type-L isozyme (L), and rabbit muscle enzyme (R). Residues making van der Waals contact with the pyridoxal moiety and the 5 -phosphate group are boxed. (From J. Biol. Chem., 261 (18), 8233 (1986)).

Previously, it has been shown that most of the residues directly interacting with AMP as well as the phosphorylatable Ser14 and its surroundings in the rabbit muscle enzyme are far less conserved in the potato type-L isozyme sequence.63 Likewise, the amino-terminal region of the potato type-H isozyme is completely different from that of the rabbit muscle enzyme over the first 80 amino acid residues, in which the sites of covalent phosphorylation and of allosteric regulation by AMP are all included. These variances in sequence are compatible with the lack of regulation in the plant phosphorylase isozymes. 

Fig. 6.4 Affinity electrophoresis of the recombinant phosphorylases from potato tuber.,3) A crude bacterial cell extract containing the type-L isozyme (lane 1), the chimeric enzyme (lane2), or the type-H isozyme (lane 3) was electrophoresed in 5% polyacrylamide gels supplemented with 0 (A), 50 (B), and 500 pg/ml (C) glycogen. After electrophoresis, the gels were stained for enzyme activity in KI-b solution. (Reproduced with permission from Goldsmith and Fletterick, Pure and Appl. Chern., 55(4), 583 (1983)).

The plant type-L isozyme is the only phosphorylase that has a large insertion in the middle of the polypeptide chain the type-H isozyme has no such insertion, like other enzymes with or without regulatory properties. The sequence containing the insertion and... 

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