Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase.

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.

Influences on transfer of selected synthetic pyrethroids from treated Formica to foods.

Children's unstructured eating habits and activities may lead to excess dietary exposures not traditionally measured by the US Environmental Protection Agency. Influence of these activities on transfer of pesticides from treated Formica to foods was studied. The objective was to perform simulation experiments using four foods (bread, apple slices, bologna, and sugar cookies) exposed to treated Formica after varied time intervals between surface contamination and contact (1, 6, and 24 h) and frequency of contact with and without recontamination. Pesticides investigated included permethrin, bifenthrin, cyfluthrin, cypermethrin, and deltamethrin. Data will be used as input parameters for transfer efficiencies (TEs) within the Children's Dietary Intake Model (CDIM), which predicts total dietary exposure of a child. Pesticide transfer from surfaces to bologna and apples was more efficient than to bread and cookies. For the bread and cookies, all pyrethroids had a TE that ranged from below detectible levels to ≤ 4%. A combined average of 32-64% and 19-43% was transferred to bologna and apples, respectively, for the three contact times for all pyrethroids. The TEs of the varied time intervals indicated that increased time between contamination and contact showed little difference for bologna, bread, and cookies, but a significant difference for apples. As long as pesticide levels are measureable on surfaces in children's eating environment, it can be concluded that transfer of pesticides to foods will take place. Foods' characteristics had an important function in the transfer of pesticides when multiple contacts occurred. Regardless of recontamination, pesticides were efficiently transferred from the treated surface to bologna. The bologna did not reach a saturation point during the contacts. Pesticides were also efficiently transferred to apples, but reached a maximum TE during the second contact. The distribution of activity factors within CDIM needs to reflect the differences in the characteristics of the foods.

BRCA1 185delAG truncation protein, BRAt, amplifies caspase-mediated apoptosis in ovarian cells.

Breast and ovarian cancer patients with germline mutations in BRCA1 respond more favorably to initial chemotherapy. We previously reported that cells from women carrying the BRCA1 185delAG founder mutation undergo an enhanced caspase-3-mediated apoptotic response. Here, we report on the transient and stable transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human ovarian surface epithelial cells and ovarian cancer cells, resulting in cells with a heterozygous background containing two BRCA1 wild-type alleles and the BRCA1 185delAG transcript. The BRCA1 185delAG truncation (BRAt) protein did not alter epithelial cell morphology or induce tumorigenesis. However, upon treatment with staurosporine, BRAt cells showed increased levels of active caspase-3 and increased cleavage of caspase-3 substrates, PARP and DFF45. Additionally, XIAP and cIAP-1 protein are at reduced levels in untreated BRAt cells as compared to control cells. BRAt also reduced levels of phosphorylated Akt and overexpression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild-type cells. Further, BRAt expression increased chemosensitivity in platinum-resistant ovarian cancer cells. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis, suggesting a possible mechanism by which ovarian cancer patients with this germline BRCA1 mutation may respond better to initial chemotherapy.

Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis.

5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.