Modification of rabbit muscle phosphorylase b by a water-soluble carbodiimide.
Glycogen phosphorylase b from rabbit muscle was rapidly inactivated by incubation with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC) at pH 5.1. The inactivation was pH-dependent and was not restored by treatment with hydroxylamine. The addition of glycine ethyl ester or N-(2,4-dinitrophenyl)-ethylenediamine (DNP-EDA) markedly increased the rate of inactivation. Of the various amino analogs of glucose tested, only glucosyl amine accelerated the inactivation, although they are all bound to the glucose 1-phosphate site of the enzyme. In the absence of amines, incorporation of about 3 mol of [metho-14C]CMC per protein monomer was observed on complete inactivation. In the presence of DNP-EDA, however, only 2 mol of [metho-14C]CMC and 1 mol of DNP-EDA were incorporated before the activity was completely lost. The treatment of phosphorylase b with CMC did not change the Km values of the enzyme for glucose 1-phosphate and AMP, in spite of the 56% inactivation. It is suggested that, in the phosphorylase-catalyzed reaction, an essential carboxyl group of the enzyme plays a role in the protonation of the glucosidic oxygen of glucose 1-phosphate.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D004136:Dinitrobenzenes / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D005944:Glucosamine / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005958:Glucosephosphates / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D006005:Phosphorylases / Q000378:metabolism*; D011817:Rabbits
D002234:Carbodiimides; D004136:Dinitrobenzenes; D005029:Ethylenediamines; D005958:Glucosephosphates; D000249:Adenosine Monophosphate; D006005:Phosphorylases; D005947:Glucose; D005944:Glucosamine; D005998:Glycine