Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.