TY - JOUR
T1 - Manganese substrate complexes of phosphofructokinase studied by pulsed magnetic resonance
AU - Cottam, G. Larry
AU - Uyeda, Kosaku
N1 - Funding Information:
This work was supported by Grant I-381 from The Robert A. Welch Foundation, Houston, Texas, a grant from the American Heart Association, Texas Affiliate, Inc., and Research Grant BM 16194f rom t,he National Institute of He&h, U. S. Public Health Service.
PY - 1973/2
Y1 - 1973/2
N2 - The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with Kd = 20 μm and ε{lunate}b ≦ 4, and perhaps, as many as 14 binding sites with Kd ~ 0.8 mm and ε{lunate}b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30-100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.
AB - The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with Kd = 20 μm and ε{lunate}b ≦ 4, and perhaps, as many as 14 binding sites with Kd ~ 0.8 mm and ε{lunate}b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30-100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.
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U2 - 10.1016/0003-9861(73)90023-4
DO - 10.1016/0003-9861(73)90023-4
M3 - Article
C2 - 4266261
AN - SCOPUS:0015578373
SN - 0003-9861
VL - 154
SP - 683
EP - 690
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -