Synthetic Evidence for the Stereochemistry of Isocitric Acid and Alloisocitric Acid Mechanism of cis-Aconitase Action

Gawron, Oscar; Glaid, Andrew J.; Lomonte, A.; Gary, Senophia

doi:10.1021/ja01554a069

Cited by 37 publications

(8 citation statements)

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“…This lack of involvement of a-methylcitrate requires a stereochemical explanation. Since (1) the reaction appears to involve carbanion intermediates which have largely the acicarboxylate form (Schloss et al, 1980), (2) the elimination is trans (Gawron et al, 1958(Gawron et al, , 1961, (3) the proton is conserved in the reaction from isocitrate to citrate (Rose & O'Connell, 1967), and (4) the hydroxyl group of citrate (and presumably isocitrate) became coordinated to a single Fe of the Fe-S cluster of aconitase (Emptage et al, 1983b), we believe the only reasonable stereochemical description of what happens in the active site is shown in Chart I.…”

Section: Discussionmentioning

confidence: 99%

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, beef liver. .alpha.-Methyl-cis-aconitate and threo-Ds-.alpha.-methylisocitrate as substrates

Schloss¹,

Emptage²,

Cleland³

1984

Biochemistry

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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)

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Section: Discussionmentioning

confidence: 99%

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, beef liver. .alpha.-Methyl-cis-aconitate and threo-Ds-.alpha.-methylisocitrate as substrates

Schloss¹,

Emptage²,

Cleland³

1984

Biochemistry

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“…Erythro and threo isomers of isocitric acid lactone were cleanly separated on Celite 535 impregnated with 0.5 N H2SO4 by the procedure of Gawron et al (1958). The lactone was shown by NMR to contain 1.00 ± 0.05 deuterium atom.…”

Section: Methodsmentioning

confidence: 99%

Isotope effect studies of the role of the metal ions in isocitrate dehydrogenase

O’Leary¹,

Limburg²

1977

Biochemistry

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Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.

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“…It was also suggested at that time that chelation of iron by substrate and groups on the enzyme might play a role in the enzymatic reaction. In the early 1960s the stereochemistry of the dehydration−hydration reaction (Scheme ) was established as occurring trans to the double bond; this was accomplished by the stereospecific synthesis of threo - d s -isocitrate, the natural substrate of the enzyme, , and was confirmed by isotope studies that provided a link to the stereochemistry of the fumarase reaction, i.e. the interconversion of malate and fumarate .…”

Section: Early Research On Aconitase (1950−1978)mentioning

confidence: 99%

Aconitase as Iron−Sulfur Protein, Enzyme, and Iron-Regulatory Protein

1996

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Synthetic Evidence for the Stereochemistry of Isocitric Acid and Alloisocitric Acid Mechanism of cis-Aconitase Action

Cited by 37 publications

References 0 publications

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, beef liver. .alpha.-Methyl-cis-aconitate and threo-Ds-.alpha.-methylisocitrate as substrates

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, beef liver. .alpha.-Methyl-cis-aconitate and threo-Ds-.alpha.-methylisocitrate as substrates

Isotope effect studies of the role of the metal ions in isocitrate dehydrogenase

Aconitase as Iron−Sulfur Protein, Enzyme, and Iron-Regulatory Protein

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