Abstract
5-Aminolevulinate synthase catalyzes the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate alpha-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 degrees C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pK(a) of 7.7 +/- 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the alpha-amino-beta-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pK(a) of 8.1 +/- 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines k(cat) and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis.
Highlights
5-Aminolevulinate synthase (ALAS)3 is a homodimeric pyridoxal 5Ј-phosphate (PLP)-dependent enzyme that is evolutionarily related to transaminases and catalyzes the first com
In the vast majority of cases the biochemical versatility of PLP can be rationalized in terms of a single property of the cofactor, the potential to act as an electron sink, and stabilize negative charge at the ␣-carbon of the substrate amino acid
The three reaction steps were visually observable in single wavelength traces of quinonoid intermediate formation and decay, as illustrated in Fig. 2, which is from a representative single turnover experiment at pH 7.5 and 30 °C
Summary
A model for catalysis by ALAS involving interconversion between “open” and “closed” conformations has been proposed based on kinetic data [19]. The structures suggest that this “loop” closes over the jugated ring system to occur the bond to be cleaved must be active site and locks succinyl-CoA in the proper juxtaposition oriented perpendicular to the plane of the PLP ring [14] This for catalysis and, subsequently, opens to allow ALA dissociaorthogonal orientation most closely aligns the sigma orbitals of tion. Alter- tor through four amino acid side chains to the thioester carnatively, decarboxylation of the reaction intermediate (IV) bonyl of succinyl-CoA (Fig. 1) This carbonyl is conserved in might initially occur through the -keto group rather than into the product ALA and appears to be required to form a quithe cofactor ring, as has been suggested for amino-oxononano- nonoid intermediate, based on the absence of a quinonoid ate synthase [18], or ALAS may represent a notable exception to upon binding of aminopentanoate, an ALA analog in which. Single turnover data were modeled using a three-kinetic-step mechanism as described by Reaction 1, whereas multiple turnover data were modeled using a twostep kinetic mechanism as described by Reaction 2
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