Characterization of the interaction of yeast enolase with polynucleotides.
Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.
al-Giery
A G
AG
Department of Biochemistry, University of Georgia, Athens 30602.
Brewer
J M
JM
D000818:Animals; D002482:Cellulose / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004277:DNA, Single-Stranded; D005988:Glyceric Acids; C008885:2-phosphoglycerate; D012313:RNA; D002482:Cellulose; D004247:DNA; D010751:Phosphopyruvate Hydratase