Conformation-reactivity relationship for pyridoxal Schiff's bases. Rates of racemization and α-hydrogen exchange of the pyridoxal Schiff's bases of amino acids

Journal of Biological Chemistry

2010

5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 Å of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K m SCoA and k cat values of the S254A variant increased, by 25-and 2-fold, respectively, the S254T substitution decreased k cat without altering K m SCoA . Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ϳ3-fold, whereas that of S254T diminished ϳ3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire ␣-oxoamine synthase family of PLP-dependent enzymes.

Section: Methodsmentioning

confidence: 99%

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

Lendrihas

Journal of Biological Chemistry

2010

“…Observed rate constants were determined by Robust Global Fitting of the acquired spectral data, using the single value decomposition software provided by OLIS, Inc. (17,18). Quality of fits was judged by analysis of the calculated residuals, and the simplest mechanism adequate to accurately describe the experimental data was used.…”

Section: Methodsmentioning

confidence: 99%

“…This orthogonal orientation most closely aligns the sigma orbitals of the bond to be cleaved with the pi orbitals of the conjugated ring system, and because these orbitals must coalesce and rehybridize in the transition state, the said orientation would theoretically provide a pathway with lowered activation energy by bringing the reactants part way along the reaction coordinate toward the transition state. The contour and magnitude of the free energy changes associated with adoption of this stereoelectronic orientation are as yet unknown, but experimental (16) and computational (17) studies have yielded estimates of 3-8 kcal/mol, corresponding to rate accelerations of up to a millionfold. In order for sequential cleavage of two of the glycine ␣-carbon bonds to occur in strict adherence with Dunathan's hypothesis, the stereochemistry involved in the ALAS mechanism described by Scheme 1 would imply torsion about the aldimine linkage of ϳ60°at some point during the time interval between formation of the two quinonoid intermediates.…”

Section: -Aminolevulinate Synthase (Alas)mentioning

confidence: 99%

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

Zhang

Journal of Biological Chemistry

2007

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...

“…Cleavage only occurs rapidly when a bond is aligned perpendicular to the plane of the PLP-aldimine conjugated system. This three-dimensional stereoelectronic orientation most closely aligns the σ-orbitals of the bond to be cleaved with the π-orbitals of the cofactor, and since these orbitals must overlap and rehybridize for any reaction to occur, the favorable positioning accelerates the rate of cleavage over the out-of-alignment α-carbon bonds, by a factor that has been estimated to be approximately a million-fold [25,26]. Thus, an important function of PLP-dependent enzymes in general is to simply bind the external aldimine complex such that the target bond is oriented perpendicular to the plane of the cofactor, thereby optimally exposing this single bond to the electron sink functionality of the cofactor, while simultaneously providing a means of discriminating against side reactions.…”

Section: Alas Reaction Chemistrymentioning

confidence: 99%

Molecular enzymology of 5-Aminolevulinate synthase, the gatekeeper of heme biosynthesis

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

2011

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.