The two phosphofructokinase gene products of Entamoeba histolytica.
Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has been described previously as a pyrophosphate (PP(i))-dependent enzyme, and the product of the second, coding for a 48-kDa protein, has been previously reported to be a PP(i)-dependent enzyme with extremely low specific activity. Here it is found that the 48-kDa protein is not a PP(i)-dependent enzyme but a highly active ATP-requiring enzyme (k(cat) = 250 s(-)1) that binds the cosubstrate fructose 6-phosphate (Fru-6-P) with relatively low affinity. This enzyme exists in concentration- and ATP-dependent tetrameric active and dimeric inactive states. Activation is achieved in the presence of nucleoside triphosphates, ADP, and PP(i), but not by AMP, P(i), or the second substrate Fru-6-P. Activation by ATP is facilitated by conditions of molecular crowding. Divalent cations are not required, and no phosphoryl transfer occurs during activation. Kinetics of the activated enzyme show cooperativity with Fru-6-P (Fru-6-P(0.5) = 3.8 mm) and inhibition by high ATP and phosphoenolpyruvate. The enzyme is active without prior activation in extracts of E. histolytica. The level of mRNA, the amount of enzyme protein, and the enzyme activity of the 48-kDa enzyme are about one-tenth that of the 60-kDa enzyme in extracts of E. histolytica trophozoites.
Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, The Chicago Medical School, North Chicago, Illinois 60064, USA.
A S
AS
Chi
Z
Z
Deng
R A
RA
Albach
R G
RG
Kemp
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology / Q000235:genetics*; D004789:Enzyme Activation; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1