TY - JOUR
T1 - Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine
AU - Pettigrew, D. W.
AU - Frieden, C.
PY - 1978
Y1 - 1978
N2 - The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme. Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25°C, with the result that regulatory kinetic behavior at pH 6.9 and 25°C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25°C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.
AB - The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme. Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25°C, with the result that regulatory kinetic behavior at pH 6.9 and 25°C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25°C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.
UR - http://www.scopus.com/inward/record.url?scp=0017822595&partnerID=8YFLogxK
M3 - Article
C2 - 148461
AN - SCOPUS:0017822595
SN - 0021-9258
VL - 253
SP - 3623
EP - 3627
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -