Transient Kinetic Studies Support Refinements to the Chemical and Kinetic Mechanisms of Aminolevulinate Synthase

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 ␣-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8 -9.2 and 5-35°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 ␣-amino-␤-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinatebound 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 pHindependent 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. 5-Aminolevulinate synthase (ALAS)3 is a homodimeric pyridoxal 5Ј-phosphate (PLP)-dependent enzyme that is evolutionarily related to transaminases and catalyzes the first committed step of tetrapyrrole synthesis in non-plant eukaryotes, as well as the ␣-subclass of purple bacteria (1-3). Many organisms, including animals and some bacteria, are known to encode two genetically distinct ALAS genes. In animals one of these genes is expressed exclusively in developing erythrocytes, and mutations in the human erythroid-specific ALAS are correlated with hereditary X-linked sideroblastic anemia, a blood disorder characterized by iron-overloaded, heme-deficient red cells (4).PLP-dependent enzymes catalyze a wide variety of reactions, including transaminations, decarboxylations, racemizations, and retro-aldol cleavages (5, 6). 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. Electrons from cleaved bonds of the covalently bound substrate can delocalize into the conjugated pyridine ring system to form quinonoid intermediates, which are often sufficiently stable to be spectroscopically observable and are characterized by strong absorption maxima of ϳ500 nm. These and other changes in the spectroscopic properties of the PLP cofactor during partial or complete reaction cycles can provide important insights into the chemical and kinetic properties of these enzymes.The generally accepted chemical me...

Targeting the Active Site Gate to Yield Hyperactive Variants of 5-Aminolevulinate Synthase

“…1). The inferred conformational dynamics of this loop are of interest because kinetic and crystallographic studies support the hypothesis that the rate of ALA (and hence protoporphyrin IX) production is controlled by opening of the active site loop coincident with product release (6,9,10). This mechanism, if correct, is notable in that the catalytic rate, which is 0.66/s at 37°C, is slow in comparison with similar conformational motions measured in other enzymes (11), suggesting that the primary selective pressures for ALAS evolution are oriented away from speed.…”

“…This approach utilizes the single value decomposition software provided by OLIS, Inc. as previously reported (9,10). The quality of the calculated fits was judged by analysis of the residuals, and the simplest mechanism that described the experimental data was used.…”

“…The determined activation energies were then used to calculate the thermodynamic activation parameters, ⌬H, ⌬G, and ⌬S, as previously described (10).…”

“…In the wild-type enzyme, single turnovers occur in three steps (supplemental Fig. S3), which have been assigned to 1) decarboxylation of a PLP-bound ␣-amino-␤-ketoadipate intermediate to form a PLP-ALA quinonoid intermediate, 2) protonation of the quinonoid intermediate to form the bound product, and 3) opening of the active site loop coincident with product released (10). Quinonoid intermediate formation for the A4 and F3 hexa-variants were increased by 9-fold (7.1 and 7.7 s Ϫ1 , respectively) compared with wild-type ALAS (0.8 s Ϫ1 ).…”

Serine 254 Enhances an Induced Fit Mechanism in Murine 5-Aminolevulinate Synthase

“…The ALAS chemical mechanism (Scheme 1) is complex and involves a high degree of stereoelectronic control, with individual steps, including the following: (i) binding of glycine; (ii) transaldimination with the active site lysine (Lys-313, murine ALAS2 numbering) to yield an external aldimine; (iii) abstraction of the pro-R proton of glycine; (iv) condensation with succinyl-CoA; (v) CoA release to generate an ␣-amino-␤-ketoadipate intermediate; (vi) decarboxylation resulting in an enolquinonoid rapid equilibrium; (vii) protonation of the enol to give an aldimine-bound molecule of ALA; and (viii) ultimately release of the product (ALA) (7). This mechanistic complexity is manifested structurally as an enzyme with an unusually high degree of sequence conservation, as exemplified by the observation that the catalytic cores of human ALAS2 and Rhodobacter capsulatus ALAS are 49% identical and 70% similar (8).…”