A general method for Ir-catalyzed asymmetric hydrogenation of tetrasubstituted α-acylpyrazole-β-alkyl cycloalkenes has been developed, furnishing 1,2- cis substituted carbo- or heterocycles with high yields and excellent enantioselectivities.
Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode γ15, a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.
Ferrochelatase catalyzes the terminal step of heme biosynthesis by inserting ferrous iron into protoporphyrin IX. The current study is aimed at understanding the structural basis of porphyrin binding and distortion in ferrochelatase-catalyzed reaction by functional analysis of a highly conserved active site loop motif. The loop was shown to contact bound porphyrin based on crystallographic and molecular modelling observations, and its role in murine ferrochelatase was assessed by random mutagenesis and steady-state kinetic analysis. To overcome the limitations of conventional kinetic assay methods for ferrochelatase, a continuous assay was developed by monitoring porphyrin fluorescence decrease using natural substrates ferrous iron and protoporphyrin IX under anaerobic conditions. For wild-type murine ferrochelatase, the assay yielded Km of 1.4 μM, Km of 1.9 μM and kcat of 4.0 min at 30 C. The results of random mutagenesis indicated that all the loop residues spanning Q248-L257 tolerated functional substitutions. While Q248, S249, G252, W256 and L257 possessed high informational content, the other five positions contained low informational content. Selected active loop variants exhibited kcat comparable to or higher than that of wild-type enzyme, while Km was increased in most variants. The kcat/Km remained largely unchanged, with
This paper reports the changes of the EEG of human bodies during saturation exposure at different depths to different mixed gases. The results of the research show that the most obvious on EEG was the appearance of diffused slow waves, usually theta waves of 4-7 times/s, and delta waves of 2-3 times/s within individual subjects. The EEG changes at 50 m were more obvious than those at 36.5 m. With the prolonging of time under high pressure, the EEG had some improvements, for instance, the slow waves decreased and the alpha waves increased. There was a certain relationship between these changes and the symptoms which appeared in the human body. The chief factor of the EEG changes is due to the effect of nitrogen narcosis during the oxygen-nitrogen diving experiment. In addition, carbon dioxide retention under the high pressure is also a factor of the EEG changes, because repeated inhaling of CO2-dense mixtures could aggravate the EEG changes and the reduction of carbon dioxide in humans by hyperventilation could improve abnormal EEGs. The main changes of the EEG during the helium-oxygen exposure at 302 m were the increase of theta waves, and even of delta waves, the decrease in alpha rhythm and the decline of amplitude of alpha waves. Increased theta index and decreased alpha index could be seen at the depth of 302 m. Under any of the above-mentioned pressure conditions when slow waves characteristic of abnormal changes appeared in the EEGs, the EEGs could be temporarily improved by photic stimulation, i.e. slow waves disappeared and alpha waves reappeared. When photic stimulation was over, alpha waves disappeared and slow waves reappeared. It was indicated that abnormal changes of the EEG under high pressure were a kind of temporary and reversible changes of the brain function.
Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248–257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (Kiapp values of 1 μM and 2.3 μM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/Km, protoporhyrin IX) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.
Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction. Functional substitutions in the 10 consecutive loop positions Gln(248)-Leu(257) were identified by genetic complementation in Escherichia coli strain Deltavis. Lys(250), Val(251), Pro(253), Val(254), and Pro(255) tolerated a variety of replacements including single substitutions and contained low informational content. Gln(248), Ser(249), Gly(252), Trp(256), and Leu(257) possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants. Selected active loop variants exhibited k(cat) values comparable with or higher than that of wild-type murine ferrochelatase. The K(m) values for porphyrin increased, except for the single mutant V251L. Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K(m) values for Fe(2+) were lowered. The k(cat)/K(m) for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y. The k(cat)/K(m) for Fe(2+) was improved. Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture. However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function.
Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode {gamma}{sub 15}, a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order ofmore » magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.« less
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