pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, and beef liver. alpha-Methyl-cis-aconitate and threo-Ds-alpha-methylisocitrate as substrates.
alpha-Methyl-cis-aconitate (cis-2-butene-1,2,3-tricarboxylate) was converted only to alpha-methylisocitrate (3-hydroxybutane-1,2,3-tricarboxylate) by aconitases from beef liver or S. lipolytica. While the kinetic parameters of beef liver (cytoplasmic) or heart (mitochondrial) aconitases did not vary over the pH range 4.9-9 with the natural substrates, and only slightly with the alpha-methyl substrates, the yeast aconitase exhibited a bell-shaped pH profile with all substrates and for binding of the competitive inhibitor, tricarballylate, with pK values around 7 and 9. The third pK of the substrates does not affect V/K, showing that these pK's are for catalytic groups on the enzyme. One of these catalytic groups presumably removes a proton to give the carbanion intermediate in the reaction, and the other protonates the hydroxyl group when it is eliminated to give water, possibly with the assistance of the Fe-S center. Beef liver aconitase showed a primary deuterium isotope effect of 1.12 (measured by equilibrium perturbation with deuterated alpha-methylisocitrate) which was pH independent and only slightly greater than the equilibrium isotope effect. Isotope effects with the yeast enzyme were also pH independent but about 1.22 on V/K (or when measured by equilibrium perturbation) and 1.7 on V. These data suggest a kinetic mechanism for beef aconitases in which product release occurs only by displacement by the substrate in a step independent of pH or of the protonation state of the substrate. With the yeast enzyme, product displacement either depends on the protonation state of the catalytic groups on the enzyme or can occur spontaneously at a finite rate.(ABSTRACT TRUNCATED AT 250 WORDS)
D000154:Aconitate Hydratase / Q000302:isolation & purification / Q000378:metabolism*; D000156:Aconitic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001203:Ascomycota / Q000201:enzymology*; D002417:Cattle; D003903:Deuterium; D017666:Deuterium Oxide; D006863:Hydrogen-Ion Concentration; D007523:Isocitrates / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D011865:Radioisotope Dilution Technique; D012442:Saccharomycopsis / Q000201:enzymology*; D013379:Substrate Specificity; D014233:Tricarboxylic Acids / Q000378:metabolism*; D014867:Water
D007523:Isocitrates; D014233:Tricarboxylic Acids; D014867:Water; C043607:alpha-methylaconitate; C021058:threo-alpha-methylisocitrate; D000156:Aconitic Acid; D003903:Deuterium; D000154:Aconitate Hydratase; D017666:Deuterium Oxide