Horticulture Fact Sheets

A series of horticulture factsheets that cover topics associated with home gardening including soil testing, soil preparation, composting, and growing onions and tomatoes

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

ABSTRACT: The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S P -enantiomers over the corresponding R P -enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k cat /K a for the R P -enantiomers of up to 2700-fold but had little effect on k cat /K a for the corresponding S P -enantiomers. The kinetic preferences for the S P -enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k cat /K a for the S P -enantiomers, while the values of k cat /K a for the R P -enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R P -enantiomers were preferred as substrates over the corresponding S P -enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site. The bacterial phosphotriesterase (PTE) 1 catalyzes the cleavage of P-O, P-F, or P-S bonds in a variety of insecticides and organophosphate nerve agents (1, 2). A generalized reaction for the hydrolysis of a simplified organophosphate substrate is illustrated in Scheme 1. Three binding pockets (small, large, and leaVing group) within the active site of PTE, which interact directly with the primary substituents attached to the phosphorus center of substrates and inhibitors, have been identified by X-ray crystallography (3). Previous investigations with chiral and achiral organophosphate triesters have shown that the reaction rates for various substrates with the wild type PTE depend to a large extent on the size and stereochemical arrangement of the substituents attached to the phosphorus core (4, 5). For example, the kinetic constants with the wild type enzyme for the two enantiomers of chiral organophosphate triesters can differ by up to 2 orders of magnitude with a clear preference for the S P -enantiomer over the R P -enantiomer (4). For the generic substrate depicted in Scheme 1, substituent Y would be bound within the large subsite while substituent X would be bound within the small subsite prior to product formation. For chiral compounds, the faster isomer is the one where substituent Y is physically larger than substituent X. An investigation into the origin of the stereoselectivity of PTE by site-directed mutagenesis has demonstrated that the kinetic preference for chiral substrates is dictated to a large extent by the size of the small subsite (6). This subsite enhances the preference for the S P -enantiomer by sterically hindering the binding and/or orientation of the R P -enantiomer to the active site (6). The most significant residues within the small subsite are Gly-60, Ile-106, Phe-132, and Ser-308. Decreasing the size of the small subsite by mutation of the single glycine residue to an alanine residue significantly increased the stereoselectivity for the preferred S P -enantiomer (6). Conversely, enlargement of the small subsite by mutation of Ile-106, Phe-132, or Ser-308 to an alanine decreased the stereoselective preference for the S P -enantiomer (6). However, enlargement of the large subsite, which prefers the bulkier substituent of the substrate during catalysis, by replacing His-254, His-257, Leu-271, or Met-317 with an alanine residue had relatively little effect on the stereose

Thermoelectric Nuclear Fuel Element Quarterly Progress Report for April- June 1960

A hot-pressing system was designed and is adaptable for bomb melting experiments up to 2000 deg C. Two devices were constructed for measuring. One was designed to measure Seebeck coefficients, resistivities, and bond resistances of swaged and machined thermoelectric wafers and the other was designed to measure thermoelectric parameters of cylindrical thermoelectric pellets up to 1000 deg C. Design studies on a fission-fired thermoelectric generator were completed. Seebeck coefficient and resistivity were determined for Li/sub .06/Ni/ sub .94/ and p type PbTe as a function of thermal fiux at 400 deg C (average). Designs of prototype thermonuclear fuel elements are presented which include single-leg, double-leg, multi-junction notched-bar, and multi-junction pelletized designs. The effects of heattreatment on the thermoelectric properties of n and p type PbTe were determined at 68 deg F. The compatibility of PbTe and GeTe with cladding materials was investigated at 510, 600, and 650 deg C. The results of life testing a swaged single-leg GeTe element are discussed. The nuclear characteristics of a 500-kw thermoelectric core that employs a rod-type fuel element were calculated. Designs of a thermoelectric reactor system that utilizes thermal circulation are presented. (For preceding period see WCAP- 1545.) (C.J.G.

Self‐pollination in island and mainland populations of the introduced hummingbird‐pollinated plant, Nicotiana glauca (Solanaceae)

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142032/1/ajb20672.pd