A-Phosphorylase

The gene for myophosphoiylase has been assigned to chromosome 1 lql3. The enzyme is a dimer of two identical 97 IcDa subunits and is the sole isoform present in skeletal muscle. Heart and brain also contain this isoform in addition to a distinct brain isoenzyme and a hybrid muscle/brain isoform. Smooth muscle also contains a phosphorylase isoform distinct from the muscle isoenzyme. If regenerating muscle fibers are present they also contain phosphorylase activity due to the presence, in fetal and developing muscle, of an isoform said to be identical with brain phosphorylase. 

Meyerhof s discovery in yeast extracts of a phosphorylase (which he named hexokinaseP) led to more detailed investigations of the specificity of this enzyme. The enzyme, which has been crystallized,89 converts D-glucose,... 

Nucleoside phosphorylases that catalyse the reversible cleavage of purine nucleosides to the free bases and ribose-1-phosphate are found in most cells, although a phosphorylase that will cleave adenosine has so far been identified only in bacteria. Recent studies have shown that ribo- and 2 -deoxyribofurano-syltransferase activity is associated with phosphorylase activity [19, 23., 222] and that both activities reside in one enzyme, which can be converted from one form to the other by substrate or product binding [20]. Upon crystallization of the enzyme from human erythrocytes a marked decrease in the ribosyl transfer reaction was observed [21b]. 

For ATP regeneration, a similar concept has been used (Berke et al, 1988). ATP can be regenerated from ADP using acetyl phosphate and the enzyme acetate kinase, upon release of acetate. The reaction is irreversible and acetyl phosphate is a relatively cheap phosphate donor. Thus, in an enzyme membrane reactor, PEGderivatized ATP was consumed by a phosphorylase or a synthetase in a reaction leading to a product of interest, and the ATP was regenerated by the acetate kinase (Figure 10.9). 

Although a true chemical synthesis of sucrose is still lacking, an enzymatic synthesis has been accomplished,34 as mentioned on page 34. Doudoroff36 was able to isolate from the bacterium Pseudomonas sac-charophila Doudoroff a phosphorylase which catalyzed the reversible reaction ... 

M17. Miller, R. M., Sies, H., Park, E. M., and Thomas, J. A., Phosphorylase and creatine kinase modification by thiol-disulfide exchange and by xanthine oxidase-initiated S-thiolation. Arch. Biochem. Biophys. 276, 355-363 (1990). 

According to Reese and Shibata,221 the biodegradative hydrolysis of galactomannan requires at least three different enzymes, namely, a-D-galactosidase, endo-/3-D-mannanase, and j3-D-mannosidase. In addition to these enzymes, there is also a report222 of a phosphorylase, identified as oligo-j8-D-mannosyl-(l— 4)-phosphorylase, which may take part in the degradation. These enzymes will now be discussed in detail. 

A phosphorylase from Escherichia coli has been purified it is specific for 2-deoxy-D-ribosyl phosphate, but can use uracil, 2-thiouracil, 5-amino-uracil, 5-bromouracil, and 2-thiothymine as a pyrimidine. Deaminases of adenosine, 2-deoxyadenosine, cytidine, and 2-deoxycytidine have been detected in Escherichia coli. 

The amino acid compositions of two phosphorylases have been determined, and are shown in Table XVI. The complete, amino acid sequence or three-dimensional structure of a phosphorylase is not yet known, and the task of delineating it will be difficult because of the large size of the molecules. However, the sequence of amino acids about an important phosphoserine residue has been reported for rabbit and human muscle phosphorylase a. In the rabbit enzyme, the sequence is... 

The maximum velocity of a phosphorylase reaction depends on the temperature and the pH, but is only obtained when the enzyme is saturated with both substrates that is, polysaccharide and inorganic phosphate for degradation, or primer and D-glucosyl phosphate for synthesis. Activities at 30° vary from 2.8 to 14 X 10 moles of D-glucosyl phosphate used up per min. per 10 g, of protein. ... 

E.D. Lowe, M.E. Noble, V.T. Skamnaki, N.G. Oikonomakos, DJ. Owen, and L.N. Johnson. 1997. The crystal structure of a phosphorylase kinase peptide substrate complex Kinase substrate recognition EMBOJ. 16 6646-6658. (PubMed)... 

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