Nuclear magnetic resonance studies of 5-aminolevulinate demonstrate multiple forms in aqueous solution

5-Aminolevulinic acid (ALA), the common precursor of heme and chlorophyll, can exist in a variety of forms at neutral pH. ¹³C NMR studies of 3-¹³C]ALA, 4-¹³C]ALA, and 5-¹³C]ALA have been used to demonstrate that the predominant species in solution under physiologic conditions is the ketone. The mole fraction of the hydrate is about 0.6%. To further substantiate the existence of the hydrate, ¹³C NMR was used to monitor ¹⁸O exchange at C₄ of 4-¹³C]ALA with H₂, ¹⁸O. Confirmation of the existence of the hydrate was achieved through direct observation by ¹H NMR. The mole fractions of the enol forms of ALA are each below 0.3%. Although direct observation of the enol forms of ALA has not been achieved, enol formation has been indirectly demonstrated by monitoring hydrogen exchange at the C₃ and C₅ methylene groups by ¹H NMR in D₂O. In neutral phosphate buffer, hydrogen exchange occurs readily at both C₃ and C₅ at a ratio of rates of 1:4. In N-trishydroxymethyl]methyl-2-aminoethanesulfonic acid-KOH buffer the hydrogen exchange rates are more than an order of magnitude slower than in phosphate buffer, but the ratio of the exchange rates remains unchanged. The results suggest that phosphate catalyzes enolization at both C₃ and C₅. To evaluate the role of the C₅ substituent in the proton exchange reactions, levulinate and 5-chlorolevulinate (5-CLA) were also monitored for proton exchange at C₃ and C₅. For levulinate, the hydrogen exchange rates in phosphate buffer are two to three orders of magnitude slower than for ALA, and the rate of hydrogen exchange at C₅ is three times slower than hydrogen exchange at C₃. The enolization rate at C₅ of 5-CLA is identical to ALA while enolization at C₃ is about threefold slower for 5-CLA than ALA. These NMR and kinetic studies suggest that under physiologic conditions, ALA rapidly equilibrates between the ketone, the hydrate at C₄, and two or more different enols (C₃---C₄ and C₄---C₅). The alternative forms of ALA may be biologically significant as active site structures for ALA synthase, glutamate semialdehyde transaminase, or porphobilinogen synthase. These NMR studies have also elucidated the structures of condensation products of ALA which can be formed under physiologic conditions. The alternative forms of ALA, as well as the autocondensation products, may serve as the active toxin in porphyrias characterized by elevated ALA levels (e.g., lead poisoning).