Type-L Isozyme

3 Structures of Phosphorylase Isozymes from Potato Tuber 6.3.1 Type-L Isozyme 

The complete amino add sequence of the potato tuber type-L isozyme was determined in this laboratory by using only protein-chemical techniques.61 65) Given the marked progress in recombinant DNA technology over the past decade, this may well be the last example of protein-chemical sequence determination for such a large protein. 

The polypeptide of type-L isozyme is composed of 916 amino acid residues. There are two covalently modified amino adds about one-fourth of the amino-terminal threonines are blocked by an acetyl group, and a lysyl residue (Lys762) is linked to the cofactor pyridoxal-P. The partial amino-terminal acetylation appeared to be a natural feature of type-L isozyme, and both the blocked and unblocked forms of type-L phosphorylase may exist in potato tubers. 

The subunit has a calculated molecular wdght of 103,916 including the acetyl group and the cofactor. The sequence is two residues longer at the amino-terminus and, similar to the bacterial enzyme, nine residues shorter at the carboxyl-terminus than the muscle enzyme when the two sequences are aligned for maximum matching (see below). Furthermore, there is a 78-residue insertion in the middle of the polypeptide chain of the type-L isozyme that occurs between residues 

The coding region of the type-L isozyme is 2898 base pairs long. It corresponds to 966 amino acid residues with a molecular weight of 109,648, and consists of two regions the amino-terminal extended sequence of 50 residues and the mature protein sequence of 916 residues. The amino acid sequence of the mature protein deduced from the cDNA structure is perfectly matched to the sequence chemically determined as described above. The stop codon, TAA, appeared at position 2942 in the cDNA sequence just after the carboxyl-terminal amino acid of the mature enzyme, indicating that no post-translational processing occurred at the carboxyl terminus. 

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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... 

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. 

Willnecker, Jahnke and Buehner96 reported recently the preliminary results of X-ray crystallographic analysis of the potato type-L isozyme at 2.6 A resolution, and showed that the overall folding of the potato phosphorylase is essentially the same as that of the rabbit muscle enzyme (more similar to the 7 -form than to the T-form). Only small differences were found in the core of the subunit, although local structures on the surface including the loops deviated significantly, being consistent with the sequence differences with some minor insertions and deletions as compared with the rabbit muscle enzyme (see above). As... 

Functional Role of the 78-Residue Insertion in Type-L Isozyme... 

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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