Ann-Sofie Johansson

Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k cat) and catalytic efficiency (k cat/K m ) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Active-site Residues Governing High Steroid Isomerase Activity in Human Glutathione Transferase A3-3

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Δ5-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 × 105-fold decrease in k cat; the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease ink cat/K m for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutationk cat/K m was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S.K i values for Δ5-androstene-3,17-dione and Δ4-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK a of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK a of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence ofk cat/K m of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK a values, 6.2 and 8.1. The basic limb of the pH dependence of k cat andk cat/K m disappears in the Y9F mutant. Therefore, the higher kinetic pK a reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.

Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases☆

Here we demonstrate that the thiol-reactive, environmentally sensitive fluorogenic molecules 6-bromoacetyl-2-dimethylaminonaphthalene and 6-acryloyl-2-dimethylaminonaphthalene are substrates for glutathione transferases (GSTs). Product formation can be measured by strong increase in fluorescence of the glutathione conjugate. As these substances display a high nonenzymatic background reaction rate, we have synthesized and characterized 6-chloroacetyl-2-dimethylaminonaphthalene, which is less reactive, favoring the enzyme-catalyzed reaction. 6-Chloroacetyl-2-dimethylaminonaphthalene was found to be a substrate for all GSTs tested. Apparent k(cat)/K(m) values (ranging between 10 and 500 mM(-1)s(-1)) revealed a strong preference for soluble GSTP1-1, GSTA1-1, and activated MGST1. Thus, 6-chloroacetyl-2-dimethylaminonaphthalene can be used in a highly sensitive assay of these GSTs. 6-Acetyl-2-dimethylaminonaphthalene derivatives are very sensitive toward solvent polarity and potentially also toward properties of binding sites in proteins. Upon binding of the conjugate to GSTs the fluorescence intensity decreased and the emission maximum was blue-shifted. Therefore the interaction of the conjugate with GSTs can be characterized with regard to both binding affinity and kinetics by stopped-flow measurements, and 6-chloroacetyl-2-dimethylaminonaphthalene can be a valuable aid in mechanistic investigations of GSTs, especially those which possess low intrinsic fluorescence.

TEM investigation of CVD graphite on nickel

Graphite films have been deposited by chemical vapour deposition from C2H4 at 1170 K on polycrystalline Ni substrates. The graphite films were characterized with respect to surface morphology and microstructure. The interface between the deposited graphite and the Ni grains was investigated by transmission electron microscopy. A smooth interface between the graphite and the Ni was observed. Electron diffraction was used to determine orientation relationships between the graphite and the Ni grains. Four different Ni grain orientations were found and for all four the graphite layers grow parallel to the Ni grain surface. A nucleation and growth mechanism involving hydrogen abstraction reactions and formation of polycyclic hydrocarbons has been proposed.

CRYSTALLINITY, MORPHOLOGY AND MICROSTRUCTURE OF CHEMICAL-VAPOR-DEPOSITED CARBON-FILMS ON DIFFERENT SUBSTRATES

Carbon films have been deposited by chemical vapor deposition from C2 H4 at 900 °C on various substrates. Film characterization with respect to crystallinity, morphology and surface microstructure was performed by X-ray diffraction, Auger electron spectroscopy, Raman spectroscopy and atomic force microscopy. The substrate material strongly affected the film characteristics. On Si, Al2O3 and quartz, turbostratic carbon was deposited, while Ni and Monel (Ni-Cu alloy) yielded highly oriented graphite. The surface microstructure on the Si, Al2O3 and quartz was quite rough, with carbon nodules 10–40 A high evenly distributed over the surface. However, the Ni substrate yielded atomically flat graphite grains separated by grain boundaries that formed ridges. The formation of ridges resulted from a higher growth rate in the grain boundaries.

Inactivation of Carcinogenic Diol Epoxides of Dibenzo[ a,l ]pyrene (Dibenzo[ def,p ]chrysene) by Human Alpha Class Glutathione Transferases

Human Alpha class glutathione transferases (hGSTs) have been incubated with the ultimate carcinogenic ( m )- anti - and (+)- syn -diol epoxides (DE) of the nonplanar dibenzo[ a,l ]pyrene (DBP). hGSTA1-1, A2-2, and A3-3 demonstrate activity with both diol epoxides ( R -absolute configuration at the benzylic oxirane carbon) whereas hGSTA4-4 virtually was inactive. (+)- syn -DBPDE was superior as substrate compared to the ( m )- anti enantiomer with hGSTA1-1 as the most efficient enzyme (k cat /K M = 464 mM m 1 s m 1 ) followed by A3-3 and A2-2 (k cat /K M = 190 mM m 1 s m 1 and 30.4 mM m 1 s m 1 , respectively). With ( m )- anti -DBPDE, the k cat /K M values were in general about 10-fold lower. Replacing ( m )- anti -DBPDE with the less complex ( m )- anti -BCDE or the less structurally distorted (+)- anti -BPDE, revealed that GSTA1-1 was 19- and 25-fold less active, respectively. hGSTA1-1 is present in human lung, a primary target tissue for PAH-induced tumors. Considering the great efficiency of this isoform relative to both Pi and Mu-class GSTs toward DBPDE, its presence and extent of expression may play a significant role in protection against this type of highly carcinogenic compounds.