title: Reaction of imidazole-citrate-deformed glycogen phosphorylase with amino acids.
abstract: Incubation of phosphorylase with L-valine in the presence of 0.4 M imidazole citrate results in a time-dependent decrease in the absorption of the enzyme-bound cofactor pyridoxal 5'-phosphate at 333 nm and the generation of a new absorption maximum at 415 nm which appears to be due to an enzyme-bound coenzyme-amino-acid aldimine adduct. Consequently, the enzyme is inactivated to less than 10% of its initial activity. The formation of the adduct of phosphorylase b with L-valine (0.1 M) proceeds with t1/2 approximately 8 min at pH 6.8 and 25 degrees C and is slightly inhibited by AMP. Phosphorylase a reacts five times more slowly than phosphorylase b. The decrease in enzymic activity is linked to the formation of the coenzyme-amino-acid adduct and is not due to resolution of the enzyme. Both the original absorption spectrum and phosphorylase activity are restored by gel filtration in the absence of L-valine and imidazole citrate. Similar reactions occur with other L-amino acids, an exception being L-cysteine which leads to resolution of the enzyme [Shaltiel, S., Hedrick, J. L. & Fischer, E. H. (1966) Biochemistry 5, 2108-2116]. No reaction is observed with D-amino acids or in the absence of imidazole citrate. Pyridoxal-reconstituted phosphorylase rapidly produces with amino acids not only the aldimine adduct but also a species absorbing at 318 nm. Enzyme-bound pyridoxal 5'-phosphate and pyridoxal exhibit a positive CD signal in the region of 333 nm; in contrast, the absorption bands of the coenzyme-amino-acid adducts at 415 nm and 318 nm are optically inactive. Neither pyridoxal-5'-phosphate-reconstituted nor pyridoxal-reconstituted phosphorylase in imidazole citrate catalyses any of the common pyridoxal-5'-phosphate-mediated reactions of amino acids, e.g. transamination, decarboxylation or racemization, thus testifying to the high degree of reaction specificity of phosphorylase.
authors.affiliation: Biochemisches Institut der Universität Zürich, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Pan
authors.forename: R
authors.initials: R
authors.lastname: Schinzel
authors.forename: D
authors.initials: D
authors.lastname: Palm
authors.forename: P
authors.initials: P
authors.lastname: Christen
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002262:Carboxy-Lyases / Q000378:metabolism; D004789:Enzyme Activation; D007093:Imidazoles / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011730:Pyridoxal / Q000378:metabolism; D011817:Rabbits; D019998:Racemases and Epimerases / Q000378:metabolism; D000637:Transaminases / Q000378:metabolism
chemical_list: D000596:Amino Acids; D007093:Imidazoles; D011730:Pyridoxal; C029899:imidazole; D006005:Phosphorylases; D000637:Transaminases; D002262:Carboxy-Lyases; D019998:Racemases and Epimerases