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G C Ferreira

Publications and source records attributed to G C Ferreira.

At least 19 recordsLinked to original sources
PubMed2005

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats.

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.

Acyl-CoA Dehydrogenase↗
PubMed2002

Mechanism of 5-aminolevulinate synthase and the role of the protein environment in controlling the cofactor chemistry.

5-Aminolevulinate synthase, a pyridoxal 5'-phosphate-dependent enzyme of the alpha-oxoamine synthase family, catalyzes the first step of the heme biosynthetic pathway in mammalian cells. This reaction entails the condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide and CoA. Mutations in the erythroid aminolevulinate synthase gene lead to a defective enzyme and are associated with the erythropoietic disorder X-linked sideroblastic anemia. In the past few years, rapid scanning-stopped-flow spectroscopy and chemical quenched-flow studies of the ALAS reaction, under single- and multi-turnover conditions, have provided important results for the interpretation of the catalytic mechanism. In particular, the role of the protein scaffold in modulating the chemical reactivity of the pyridoxal 5'-phosphate cofactor and, thus, the catalytic pathway of ALAS has been investigated in our laboratory using transient kinetics and global analysis of the kinetic data.

5-Aminolevulinate Synthetase↗
PubMed2001

The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase.

The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and (1)H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H(2)O solutions and micellar environments. A well defined amphipathic alpha-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short alpha-helix, defined by A5-L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and (1)H NMR study of hemin and psALAS indicated that a heme-peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.

5-Aminolevulinate Synthetase↗
PubMed2001

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants.

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn(2+) results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

Alanine↗
PubMed2001

Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

5-Aminolevulinate Synthetase↗
PubMed2000

Mutations in the iron-sulfur cluster ligands of the human ferrochelatase lead to erythropoietic protoporphyria.

Ferrochelatase (FECH; EC 4.99.1.1) catalyzes the terminal step of the heme biosynthetic pathway. Defects in the human FECH gene may lead to erythropoietic protoporphyria (EPP), a rare inherited disorder characterized by diminished FECH activity with protoporphyrin overproduction and subsequent skin photosensitivity and in rare cases liver failure. Inheritance of EPP appeared to be autosomal dominant with possible modulation by low expression of the wild-type FECH allele. Animal FECHs have been demonstrated to be [2Fe-2S] cluster-containing proteins. Although enzymatic activity and stability of the protein appear to be dependent on the presence of the [2Fe-2S] cluster, the physiologic role of the iron-sulfur center remains to be unequivocally established. Three of the 4 [2Fe-2S] cluster-coordinating cysteines (ie, C403, C406, and C411 in the human enzyme) are located within the C-terminal domain. In this study 5 new mutations are identified in patients with EPP. Three of the point mutations, in 3 patients, resulted in FECH variants with 2 of the [2Fe-2S] cluster cysteines substituted with tyrosine, serine, and glycine (ie, C406Y, C406S, and C411G) and with undetectable enzymatic activity. Further, one of the patients exhibited a triple point mutation (T(1224)-->A, C(1225)-->T, and T(1231)-->G) leading to the N408K/P409S/C411G variant. This finding is entirely novel and has not been reported in EPP. The mutations of the codons for 2 of the [2Fe-2S] cluster ligands in patients with EPP supports the importance of the iron-sulfur center for the proper functioning of mammalian FECH and, in at least humans, its absence has a direct clinical impact. (Blood. 2000;96:1545-1549)

Female↗
PubMed2000

Transcriptional regulation of the murine erythroid-specific 5-aminolevulinate synthase gene.

5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway in mammalian cells. Separate genes encode the two isoforms: ubiquitously expressed ALAS (ALAS1) and erythroid-specific ALAS (ALAS2). Transcription of the ALAS2 gene is only activated during erythroid cell differentiation. This stimulation allows for the formation of hemoglobin-specific heme. The 5'-flanking region of the mouse ALAS2 gene was studied in order to define its erythroid-specific function in transcriptional activation. Putative binding sites for the erythroid-specific nuclear factors GATA-1, NF-E2, and EKLF were identified within the first 300bp region of the mouse ALAS2 5'-flanking region. However, this 300bp region alone did not efficiently activate transient expression in erythroid MEL and K562 cell lines. Additional DNA regulatory sequences found within 300-718bp upstream of the transcription start site were required for maximal transcriptional activation, even though these regions stimulated similar expression in the non-erythroid HeLa and NIH/3T3 cells. This suggests that cis-acting elements present in the 5'-flanking region are not responsible for maintenance of transcriptional silencing in non-erythroid cell lines and that tissue-specific regulation of ALAS2 depends on other regions of the gene or on chromatin remodeling. A putative hypoxia inducible factor 1 (HIF-1) response element was identified within the 300-718bp upstream region. Significantly, two proximal GATA-1-binding sites (-118/-113 and -98/-93) and a region located within -518 to -315bp of the mouse ALAS2 promoter were essential for transcriptional activation during chemically induced differentiation of MEL cells, implying their importance in conferring erythroid specificity to the ALAS2 transcriptional activation. This is the first study to delimit the cis-acting region responsible for activation of the ALAS2 promoter upon dimethyl-sulfoxide induction in MEL cells.

3T3 Cells↗
PubMed2000

Porphyrin interactions with wild-type and mutant mouse ferrochelatase.

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe(2+) chelation into protoporphyrin IX. Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. On the basis of changes in the UV-vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is substoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive lines and the vinyl vibrational mode in the resonance Raman spectra. Shifts in the resonance Raman lines of free-base and metalated porphyrins bound to the wild-type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle. However, the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decompositions indicate a homogeneous ruffled deformation for the nickel protoporphyrin bound to the wild-type ferrochelatase, whereas both planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free-base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together, these findings suggest that distortion occurs in murine ferrochelatase for some porphyrins, even without metal binding, which is apparently required for the yeast ferrochelatase.

Amino Acid Substitution↗
PubMed1999

Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release.

5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.

5-Aminolevulinate Synthetase↗
PubMed1999

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates.

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.

5-Aminolevulinate Synthetase↗
PubMed1999

Ferrochelatase.

Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX. It is encoded by a single gene, and mutations in the human gene are associated with the inherited disorder, erythropoietic protoporphyria. With the development of heterologous overexpression systems and the ready availability of recombinant ferrochelatase, new structural elements have been identified and new aspects of the ferrochelatase-catalyzed reaction mechanism have been unraveled. Namely, a [2Fe-2S] cluster is a prosthetic group in mammalian ferrochelatase, a conserved and essential histidine residue appears to be involved in the binding of the metal substrate and a conserved glutamate residue has been proposed to have a catalytic role. The three-dimensional structure for Bacillus subtilis ferrochelatase, the only known 'water-soluble' ferrochelatase, revealed that the protein contains two similar domains, each of which has a four-stranded beta-sheet flanked by alpha-helices; the active site was modeled to be in a cleft defined by the two domains. The definition of the structure and catalytic mechanism of ferrochelatase should help in the interpretation of the impact caused by erythropoietic porphyria mutations.

Animals↗
PubMed1998

Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor.

5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.

5-Aminolevulinate Synthetase↗
PubMed1998

Role of arginine 439 in substrate binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes. Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group. To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis. The R439K mutant retained 77% of the wild-type activity; its K(m) values for both substrates increased 9-13-fold, while the activity of R433K increased 2-fold and the K(m) values for both substrates remained unchanged. R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay. In contrast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, while Kd values for glycine for both R433K and R433L mutants were similar to those of the wild-type. However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase in K(d)methylamine for R439L. R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 8.3 degrees C lower than that of the wild-type enzyme. In addition, in vivo complementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149. Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.

5-Aminolevulinate Synthetase↗
PubMed1998

The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.

5-Aminolevulinate Synthetase↗
PubMed1996

Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and some bacteria. The enzyme belongs to a large family of enzymes that use pyridoxal 5'-phosphate as an essential cofactor. We previously analyzed the informational content contained in each residue of a conserved glycine loop, which we proposed to form part of the cofactor binding site [Gong, J., & Ferreira, G. C. (1995) Biochemistry 34, 1678-1685]. We found that Gly-142 and -144 contain high informational content, and we identified G144A, G144S, G144T, and G142C as functional mutants. Here, the catalytic parameters, cofactor affinities, and spectral and thermostability properties of these four glycine mutants are determined to examine the function of the glycine loop. In addition, computer models of the glycine loops from the wild-type and mutant enzymes were generated, using glycogen phosphorylase b as the structural template. G144A, G144S, G144T, and G142C displayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and 15-fold increases, respectively, in the dissociation constant value for binding of the cofactor. While the turnover numbers for G144A, G144S, G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value, respectively, the K(m) values for both substrates remained unchanged, with the exception of the G142C K(m)Gly, which showed a 4-fold increase. The UV-visible and CD spectra of Gly-144 mutants were similar to those of the wild type; however, the spectral properties of G142C suggest that this mutant binds the cofactor in a different mode at the active site. G144A, G144S, G144T, and G142C were also found to be less stable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 3.5, 3, 3.5, and 5 degrees C, respectively, lower than that of the wild-type enzyme. Collectively, computer modeling of the wild-type and mutant forms of the ALAS glycine loop and biochemical and spectroscopic characterization of G144A, G144S, G144T, and G142C strongly suggest that the conserved glycine loop in 5-aminolevulinate synthase is a pyridoxal 5'-phosphate cofactor binding motif.

5-Aminolevulinate Synthetase↗
PubMed1996

Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation.

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sci. 2, 1959-1965], while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function [Gong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E. coli strain acquired heme prototrophy. The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits.

5-Aminolevulinate Synthetase↗
PubMed1996

Poisoning of wildlife in South Africa.

Over a period of 7 years the Toxicology Diagnostic Laboratory at the Onderstepoort Veterinary Institute investigated 370 suspected incidents of poisoning of wildlife. A diagnosis was made in 166 of the outbreaks. Synthetic organic pesticides were responsible for 82.5% of the poisonings. More than 90% of the poisonings were maliciously intended and monocrotophos was the chemical most often used. The data were derived from authenticated outbreaks of poisoning and indicate the chemicals most often incriminated in acute poisoning of wildlife. Of all wild animals, birds are the most frequent victims of poisoning. Uncommon toxicities such as ionophore, cardiac glycoside poisoning and chronic fluorosis are also described.

Animal Diseases↗