Pyridoxal-5'-phosphate (PLP) is an obligatory cofactor for the homodimeric mitochondrial enzyme 5-aminolevulinate synthase (ALAS), which controls metabolic flux into the porphyrin biosynthetic pathway in animals, fungi, and the α-subclass of proteobacteria. Recent work has provided an explanation for how this enzyme can utilize PLP to catalyze the mechanistically unusual cleavage of not one but two substrate amino acid α-carbon bonds, without violating the theory of stereoelectronic control of PLP reaction-type specificity. Ironically, the complex chemistry is kinetically insignificant, and it is the movement of an active site loop that defines kcat and ultimately, the rate of porphyrin biosynthesis. The kinetic behavior of the enzyme is consistent with an equilibrium ordered induced-fit mechanism wherein glycine must bind first and a portion of the intrinsic binding energy with succinyl-Coenzyme A is then utilized to perturb the enzyme conformational equilibrium towards a closed state wherein catalysis occurs. Return to the open conformation, coincident with ALA dissociation, is the slowest step of the reaction cycle. A diverse variety of loop mutations have been associated with hyperactivity, suggesting the enzyme has evolved to be purposefully slow, perhaps as a means to allow for rapid up-regulation of activity in response to an as yet undiscovered allosteric type effector. Recently it was discovered that human erythroid ALAS mutations can be associated with two very different diseases. Mutations that down-regulate activity can lead to X-linked sideroblastic anemia, which is characterized by abnormally high iron levels in mitochondria, while mutations that up-regulate activity are associated with X-linked dominant protoporphyria, which in contrast is phenotypically identified by abnormally high porphyrin levels. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

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...

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k cat and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu 2؉ > Zn 2؉ > Co 2؉ > Ni 2؉ and can be alleviated by the addition of metal complexing agents such as ␤-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

The rate of porphyrin biosynthesis in mammals is controlled by the activity of the pyridoxal 5-phosphate-dependent enzyme 5-aminolevulinate synthase (EC 2.3.1.37). Based on the postulate that turnover in this enzyme is controlled by conformational dynamics associated with a highly conserved active site loop, we constructed a variant library by targeting imperfectly conserved noncatalytic loop residues and examined the effects on product and porphyrin production. Functional loop variants of the enzyme were isolated via genetic complementation in Escherichia coli strain HU227. Colony porphyrin fluorescence varied widely when bacterial cells harboring the loop variants were grown on inductive media; this facilitated identification of clones encoding unusually active enzyme variants. Nine loop variants leading to high in vivo porphyrin production were purified and characterized kinetically. Steady state catalytic efficiencies for the two substrates were increased by up to 100-fold. Presteady state single turnover reaction data indicated that the second step of quinonoid intermediate decay, previously assigned as reaction rate-limiting, was specifically accelerated such that in three of the variants this step was no longer kinetically significant. Overall, our data support the postulate that the active site loop controls the rate of product and porphyrin production in vivo and suggest the possibility of an as yet undiscovered means of allosteric regulation. Aminolevulinate (ALA)2 is the universal building block of tetrapyrolle biosynthesis (1). In nonplant eukaryotes and the ␣-subclass of purple bacteria, the production of ALA is catalyzed by the pyridoxal 5Ј-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS) (EC 2.3.1.37), in a reaction involving the Claisen-like condensation of succinyl-coenzyme-A and glycine to yield CoA, carbon dioxide, and ALA (2). ALAS catalyzes the first committed step of tetrapyrrole biosynthesis in these organisms, which is also the rate-determining step of the pathway. Consequently, overexpression of ALAS in prokaryotic and eukaryotic cells results in accumulation of the photosensitizing heme precursor protoporphyrin IX (3). This property could potentially lead to novel applications of ALAS or ALAS variants in photodynamic therapy of tumors and other dysplasias (4).ALAS is classified as a fold-type I PLP-dependent enzyme and, like the evolutionarily related L-amino acid transaminases (5), functions as a homodimer wherein a PLP cofactor is bound at each of the two active sites, which are recessed in clefts at the subunit interface (6, 7). X-ray crystal structures of ALAS from Rhodobacter capsulatus and the closely related enzyme 8-amino-7-oxononanoate synthase from Escherichia coli reveal an induced fit type mechanism wherein binding of substrates and product, respectively, trigger closure of an extended loop over the active site (6, 8) (Fig. 1). The inferred conformational dynamics of this loop are of interest because kinetic and crystallographic studies support the hypothes...

5-Aminolevulinate synthase (ALAS) is