Involvement and identification of a lysine in the PPi-site of pyrophosphate-dependent phosphofructokinase from Giardia lamblia.
The substrate binding and/or catalytic site of the pyrophosphate-dependent phosphofructokinase (PPi-PFK) of Giardia lamblia was investigated using an ATP affinity label, 2',3'-dialdehyde of ATP, oxidized ATP (oATP), for the involvement of lysine residues. The enzyme, which uses PPi rather than ATP as a substrate was inhibited by low concentrations of oATP. Oxidized ATP behaves as an affinity label for the substrate binding site as evidenced by saturation kinetics with the formation of reversible complex prior to inactivation, and the observation that the inactivation was stoichiometric with the amount of oATP incorporated which extrapolated to 1 mol per mol of monomeric PPi-PFK. The critical lysine modified by oATP is proposed to be located at the PPi-binding site since complete protection is afforded by PPi; and under steady-state, PPi was competitive with the inhibitor. Other substrates of the reaction in either the forward or reverse direction did not completely protect against inactivation. This is further confirmed by the non-competitive inhibition displayed by either Pi or fructose 1,6, bisphosphate. Furthermore, the Km values for Pi and fructose 1,6 bisphosphate of the oATP-modified enzyme were not altered. The oATP-modified peptides were analyzed by HPLC peptide mapping, and the profile showed a major peak absorbing at 258 nm, which was absent when the modification was carried out in the presence of MgPPi. This peptide was sequenced and found to contain Lys-497. These results suggest that the essential lysine-497 modified by oATP is involved in the binding and/or catalysis of PPi and that an ATP-type of binding domain, with reference to the phosphoryl groups, is present in the PPi-dependent phosphofructokinase of Giardia.
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Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4983, USA.
Phillips
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005635:Fructosediphosphates / Q000378:metabolism; D016829:Giardia lamblia / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000345:Affinity Labels; D005635:Fructosediphosphates; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D008239:Lysine