Identification of highly reactive cysteinyl and methionyl residues of rabbit muscle phosphofructokinase.
The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.
D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D000242:Cyclic AMP / Q000494:pharmacology; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology; D005636:Fructosephosphates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008715:Methionine; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011817:Rabbits; D013329:Structure-Activity Relationship; D014357:Trypsin
D005636:Fructosephosphates; D007461:Iodoacetates; D010446:Peptide Fragments; C027618:fructose-6-phosphate; D008715:Methionine; D000242:Cyclic AMP; D010732:Phosphofructokinase-1; D014357:Trypsin; D003545:Cysteine; D019807:Iodoacetic Acid