RECONSTITUTION OF APOPHOSPHORYLASE WITH PYRIDOXAL 5'-PHOSPHATE ANALOGS

The purpose of this study was to probe the catalytic role of pyridoxal 5′-phosphate in muscle glycogen phosphorylase (EC 2.4.1.1). Pure apophosphorylases b and a were reacted with analogs of pyridoxal 5′-phosphate modified in every position around the pyridine ring and the enzymaticactivity and state of aggregation of the system were investigated. Slight reactivations observed with high concentrations of pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate couldbe ascribed to trace contamination by pyridoxal 5′- phosphate. Restoration of enzymatic activity was observed with analogs modified in positions 2,3,4, and 6. Removal of the methyl group in position 2 (2-norpyridoxal 5′-phosphate) gave 65 % reactivation while the bulkier 2-ethyl derivative did not reactivate the apoenzyme. 3-O-Methylpyridoxal 5′-phosphate activated apophosphorylases b and a 25 and 40%, respectively. None of the analogs modified inposition4 restored enzymatic activity, indicating that a 4-formyl group is required for the bindingof the cofactor. Once bound, however, the formyl group can be modified, e.g., by sodium borohydride reduction that covalently fixes the cofactor to the protein with retention of activity. The 6-methyl derivative was also active. Substituion of the pyridine nitrogen and all replacements of the 5 ′-phosphate group led to inactive products. It is concluded that these two groups, in addition to the 4-formyl group, are required for the binding of the cofactor or enzymatic activity or both. In all instances, restoration of activity was accompanied by restoration of the state of aggregation of the enzyme. However, reaggregation in itself did not confer catalytic activity on the protein since several analogs that restored the quaternary structure of the enzyme yielded inactive products. © 1969, American Chemical Society. All rights reserved.