The activity of a Zn(II) complex of a tetradentate, tripodal ligand for catalyzing phosphodiester cleavage is enhanced 750-fold by introducing three hydrogen bond donors to the ligand. Inhibition studies show that the Zn-aqua complex is the kinetically active form and that it binds the transition state with a formal dissociation constant of 3 x 108 M-1. The effect of these ligand modifications on the transition-state affinity is comparable to the rate acceleration provided by the metal ion itself. Overall, this mononuclear complex is more active than the most reactive dinuclear Zn(II) complexes reported to date.

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. ␤-Phosphoglucomutase catalyses the isomerization of ␤-glucose-1-phosphate to ␤-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19 F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF 3 ؊ mimics the transferring PO 3 ؊ moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of ␤-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.enzyme mechanism ͉ fluoride inhibition ͉ NMR structure ͉ phosphoryl transfer ͉ isosteric isoelectronic ͉ transition state analogue P hosphate transfer reactions play a central role in metabolism, regulation, energy housekeeping and signaling (1). As phosphate esters are kinetically extremely stable, efficient catalysis is crucial for the control of these cellular processes. Although model studies have taught us much about the intrinsic chemical mechanisms (2), our understanding of the origins of the enormous enzymatic rate accelerations involved, up to a factor of 10 21 (3), is far from complete (4). A snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed (5). However, is this realistic given how elusive high-energy intermediates and transition states (TSs) inevitably are? The direct observation of TSs for simple organic reactions has required ultrafast lasers with femtosecond resolution (6) and no physical or spectroscopic method is available to observe the structure of TSs of enzymatic reactions directly. Thus transition state analogues that bind tightly in an enzyme active site have been of paramount importance in defining the structural and energetic framework for catalysis (7,8).An observation that appears to challenge this paradigm arises from structural studies with ␤-phosphoglucomutase (␤-PGM, EC 5.4.2.6): namely, that a high-energy phosphorane on the reaction pathway has been observed directly by x-ray crystallography, demonstrating how the enzyme interacts with a very high-energy, metastable species (9). The latter also apparently demonstrated that the enzyme catalyzed reaction proceeds through an addition-elimination mechanism, a reaction pathway not observed in solution for phosphate monoester anions. However, the observation of an enzyme "caught in the act" is surprising: the demands of turnover mean that the enzyme would gain no apparent advant...

Recently, the fluorescent detection of carbon monoxide (CO) in living cells has attracted great attention. However, due to the lack of effective ways to construct fluorescent CO probes, fluorescent detection of CO in living cells is still in its infancy. In this paper, we report for the first time the use of allyl ether as a reaction site for construction of fluorescent CO probes. By this way, two readily available allyl fluorescein ethers were prepared, which were found to be highly selective and sensitive probes for CO in the presence of PdCl. These probes have the merits of good stability, good water-solubility, and rapid and distinct colorimetric and remarkable fluorescent turn-on signal changes. Moreover, a very low dose of these two probes can be used to detect and track CO in living cells, indicating that these two probes could be very promising biological tools for CO detection in living systems. Overall, this work provided not only two new promising fluorescent CO probes but also a new way to devise fluorescent CO probes.

Summary• The growth of a plant organ to its characteristic size is regulated by an elaborate developmental program involving both internal and external signals. Here, we identify a novel Arabidopsis gene, ORGAN SIZE RELATED1 (OSR1), that is involved in regulation of organ growth and overall organ size.• A combination of genetic, cytological and molecular approaches was used to characterize the expression profile, subcellular localization and roles of OSR1 during organ growth.• Ectopic expression of OSR1 in Arabidopsis resulted in enlarged organs, as a consequence of increases in both cell number and cell size. OSR1 shares a conserved OSR domain with ARGOS and ARGOS-LIKE (ARL), which is sufficient for their functions in promoting organ growth. OSR1 is a plant hormone-responsive gene and appears to act redundantly with ARGOS and ARL during organ growth. The OSR proteins are localized to the endoplasmic reticulum.• Our results suggest that three co-evolved members of the OSR family may act coordinately to orchestrate growth signals and cell proliferation and expansion, thereby affecting organ growth and final organ size.