Can one predict protein stability? An attempt to do so for residue 133 of T4 lysozyme using a combination of free energy derivatives, PROFEC, and free energy perturbation methods.

Free energy derivatives, pictorial representation of free energy changes (PROFEC) and free energy perturbation methods were employed to suggest the modifications that may improve the stability of a mutant T4 lysozyme with a S-2-amino-3-cyclopentylpropanoic acid residue (Cpe) at position 133. The free energy derivatives and PROFEC methods were used to locate promising sites where modifications may be introduced. The effects of several candidate modifications on the enzyme's stability were analyzed by the free energy perturbation method. We found that this scheme is able to effectively suggest modifications that may increase the enzyme's stability. The modifications investigated are the introduction of a methyl, a tert-butyl or a trifluoromethyl group at the Cepsilon2 position and a cyclopropyl group between the Cdelta2 and Cepsilon2 position on the cyclopentyl ring. The stereochemistry of the introduced groups (in the alpha or beta configurations) was studied. Our calculations predict that the introduction of a methyl group in the alpha configuration or a cyclopropyl group in the beta configuration will increase the stability of the enzyme; the introduction of the two groups in the other configurations and the other modifications will decrease the stability of the enzyme. The results indicate that packing interactions can strongly influence the stability of the enzyme.

The application of three approximate free energy calculations methods to structure based ligand design: trypsin and its complex with inhibitors.

Three approximate free energy calculation methods are examined and applied to an example ligand design problem. The first of the methods uses a single simulation to estimate the relative binding free energies for related ligands that are not simulated. The second method is similar, except that it uses only first derivatives of free energy with respect to atomic parameters (most often charge, van der Waals equilibrium distance, and van der Waals well depth) to calculate free energy differences. The last method PROFEC (Pictorial Representation of Free Energy Components), generates contour maps that show how binding free energy changes when additional particles are added near the ligand. These three methods are applied to a benzamidine/trypsin complex. They each reproduce the general trends in the binding free energies, indicating that they might be useful for suggesting how ligands could be modified to improve binding and, consequently, useful in structure-based drug design.

Photosynthetic o(2) exchange kinetics in isolated soybean cells.

Light-dependent O(2) exchange was measured in intact, isolated soybean (Glycine max. var. Williams) cells using isotopically labeled O(2) and a mass spectrometer. The dependence of O(2) exchange on O(2) and CO(2) was investigated at high light in coupled and uncoupled cells. With coupled cells at high O(2), O(2) evolution followed similar kinetics at high and low CO(2). Steady-state rates of O(2) uptake were insignificant at high CO(2), but progressively increased with decreasing CO(2). At low CO(2), steady-state rates of O(2) uptake were 50% to 70% of the maximum CO(2)-supported rates of O(2) evolution. These high rates of O(2) uptake exceeded the maximum rate of O(2) reduction determined in uncoupled cells, suggesting the occurrence of another light-induced O(2)-uptake process (i.e. photorespiration).Rates of O(2) exchange in uncoupled cells were half-saturated at 7% to 8% O(2). Initial rates (during induction) of O(2) exchange in uninhibited cells were also half-saturated at 7% to 8% O(2). In contrast, steady-state rates of O(2) evolution and O(2) uptake (at low CO(2)) were half-saturated at 18% to 20% O(2). O(2) uptake was significantly suppressed in the presence of nitrate, suggesting that nitrate and/or nitrite can compete with O(2) for photoreductant.These results suggest that two mechanisms (O(2) reduction and photorespiration) are responsible for the light-dependent O(2) uptake observed in uninhibited cells under CO(2)-limiting conditions. The relative contribution of each process to the rate of O(2) uptake appears to be dependent on the O(2) level. At high O(2) concentrations (>/=40%), photorespiration is the major O(2)-consuming process. At lower (ambient) O(2) concentrations (

Rate-temperature curves as an unambiguous indicator of biological activity in soil.

Experiments are described in which we used a mass spectrometer to monitor O(2) uptake of enclosed soil samples as a function of temperature. We found that an Arrhenius plot of the rate of O(2) uptake showed pronounced local maxima attributable to biological activity, whereas similar plots of rates obtained with abiotic soils yielded straight lines. This procedure thus provides a basis for distinguishing biological from chemical activity for reactions, such as O(2) uptake, that can occur via either biological or chemical pathways.

Photoreduction of O(2) Primes and Replaces CO(2) Assimilation.

A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O(2) evolution or CO(2) uptake was observed, O(2) evolution was normal but completely compensated by O(2) uptake. Similarly, when CO(2) uptake was totally or partially inhibited, O(2) evolution proceeded at a high (near maximal) rate. Under all conditions, O(2) uptake balanced that fraction of the O(2) evolution which could not be accounted for by CO(2) uptake.From these observations we concluded that O(2) and CO(2) are in direct competition for photosynthetically generated reducing power, with O(2) being the main electron acceptor during the induction process and under other conditions in which CO(2) reduction cannot keep pace with O(2) evolution. The high rate of the O(2) uptake reaction observed in the presence of iodoacetamide, KCN, or carbonyl cyanide p-trifluoromethyoxyphenylhydrazone, suggests that a special high capacity oxidase distinct from ribulose diphosphate oxygenase exists in whole cells. The rapid reduction of molecular O(2) after a period of darkness probably serves as a priming reaction for the photosynthetic apparatus. The high steady state rate of the O(2) cycle in the absence of CO(2) fixation suggests that the regulation of photosynthesis does not involve significant changes in the rate of photochemical electron transport.

(Minus) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, an enzyme in the biosynthetic pathway of chlorophyll in Zea mays.

The enzyme (-) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (-) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays. Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin. THE FOLLOWING SCHEME OF CHLOROPHYLL SYNTHESIS IN HIGHER PLANTS IS PROPOSED: delta-aminolevulinic acid --> --> --> protoporphyrin --> magnesium protoporphyrin --> magnesium protoporphyrin monomethyl ester --> --> --> chlorophyll a.