Interaction of rabbit muscle aldolase at high ionic strengths with vanadate and other oxoanions.
Reductive, nonreductive, and photolytic interactions of vanadate with fructose-1,6-bisphosphate aldolase were examined and used to explore the interactions of oxoanions with aldolase. Aldolase is known to interact strongly with oxoanions at low ionic strength and weakly at higher ionic strength. Oxoanions inhibit aldolase competitively with respect to fructose 1,6-bisphosphate although the location of the oxoanion binding site on aldolase remains elusive. In this work, the interaction of aldolase with a series of oxoanions was compared at ionic strength approaching physiologic levels. The size and shape of the anion were important for the effective binding to aldolase, and no significant increase in affinity for aldolase was observed by the addition of alkyl groups to the oxoanions. Vanadate competitively inhibits aldolase in a manner analogous to the other oxoanions. Since vanadate solutions contain a mixture of vanadate oxoanions, the nature of the inhibition was determined using a combination of enzyme kinetics and 51V NMR spectroscopy. Aldolase contains a significant number of thiol functionalities, and as expected, vanadate undergoes redox chemistry with them, generating an irreversibly inhibited aldolase. This oxidative chemistry was attributed to the vanadate tetramer, whereas vanadate dimer was a reversible inhibitor. Vanadate monomer does not significantly interact with aldolase reversibly or irreversibly. Vanadyl cation has the lowest inhibition constant under these high ionic strength conditions. Using Yonetani-Theorell analysis, it appears that phosphate, pyrophosphate, and sulfate bind to the same site on aldolase, whereas vanadate, arsenate, and molybdate bind to another site. UV light-induced photocleavage of aldolase by vanadate was examined, and the loss of aldolase activity was correlated with cleavage of the aldolase subunit. Further studies using vanadium as a probe should reveal details on the location of the vanadate and vanadyl cation binding sites. This study suggests several sites on aldolase will accommodate oxoanions, and one of these sites also accommodates vanadyl cation.
Crans
D C
DC
Department of Chemistry, Colorado State University, Fort Collins 80523.
Sudhakar
K
K
Zamborelli
T J
TJ
D000818:Animals; D000838:Anions; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008962:Models, Theoretical; D009132:Muscles / Q000201:enzymology*; D009994:Osmolar Concentration; D011817:Rabbits; D014638:Vanadates / Q000378:metabolism*
D000838:Anions; D014638:Vanadates; D005634:Fructose-Bisphosphate Aldolase