Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity.
The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.
Zographos
S E
SE
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece.
Oikonomakos
N G
NG
Dixon
H B
HB
Griffin
W G
WG
Johnson
L N
LN
Leonidas
D D
DD
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D005958:Glucosephosphates / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology*; D010763:Phosphorylase b / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013431:Sulfates / Q000494:pharmacology*
D005958:Glucosephosphates; D007527:Isoenzymes; D013431:Sulfates; D000249:Adenosine Monophosphate; C031590:glucose-1-phosphate; D010763:Phosphorylase b