Cover story

The Michaelis-Menten equation, first reported in 1913, has become an indispensable formalism for understanding enzyme kinetics. Traditionally, Michaelis-Menten kinetics has been measured in 'bulk' solution. However, these en masse measurements may overlook important features of catalysis by individual enzyme molecules. To investigate whether the Michaelis-Menten formalism holds at the single-molecule level, Xie and co-workers visualized enzymatic turnover of immobilized β-galactosidase using single-molecule fluorescence microscopy. The authors demonstrated that, over time, the behavior of each molecule recapitulates Michaelis-Menten kinetics. However, for individual enzymes under high concentrations of substrate, enzyme activity showed a 'memory effect', which was attributed to conformational heterogeneity among individual molecules. The study suggests that confirmational variations among individual enzymes could be important in cells, where only a few copies of each enzyme may be present. [Articles, p. 87 ; News & Views, p. 66 ] TLS

Bcr-abl inhibition a new way

BCR-ABL is a gene fusion often generated in chronic myelogenous leukemia (CML). The resulting fusion protein has constitutive kinase activity that plays a causative role in CML. The successful small-molecule drug imatinib (STI571, Gleevec; Novartis Pharma) inhibits Bcr-abl kinase activity, validating it as a cancer therapeutic target. In later stages of the disease, resistance rapidly arises to imatinib, which has led to the need for new therapeutics. By using a differential cytotoxicity screen, Gray and colleagues identified a class of 4,6-disubstituted pyrimidine compounds that successfully inhibited proliferation of Bcr-abl–expressing cells with little toxicity in Bcr-abl–negative cells. These small molecules appeared to target Bcr-abl kinase activity directly; however, the small molecules had no effect on the protein's activity in vitro, suggesting a mechanism of action that, unlike that of imatinib, does not target the active site. These compounds appear selective for Bcr-abl and have limited toxicity, which makes them promising leads for next-generation CML therapeutics. [Articles, p. 95 ; News & Views, p. 63 ] JK

Sneaking into DNA

Inside cells DNA is wrapped around histones to form the nucleosome core particle (NCP). Packaging it in this way serves to make the DNA compact enough to fit in the nucleus and to help regulate access to DNA. Based on the static structure of the NCP, some parts of DNA would appear inaccessible to small molecules and proteins. Two natural products with potent antitumor activity, duocarmycin SA and yatakemycin, function by alkylating DNA. Although they are known to function in cells, it was not clear which stretches of DNA would be targeted in the NCP as compared to 'free' DNA. Boger and colleagues have now shown that duocarmycin SA and yatakemycin both alkylate NCP DNA with the same efficiency and sequence preferences as unbound DNA. This is even true for sites that appear inaccessible in the NCP structure. These results highlight the dynamic nature of the NCP and also provide important evidence for the relevance of in vitro studies to the in vivo action of these natural products. [Letters, p. 79 ; News & Views, p. 64 ] JK

Watching actin dance

The cytoskeleton allows cells to change shape and move in a directed manner, which is required for cell motility and muscle contraction. Filamentous actin (F-actin) is a major component of the cytoskeleton. The structure of F-actin can change spontaneously, but it can also be controlled by association with motor proteins, including myosin, that walk along F-actin during cellular movement. To investigate the molecular basis of actin-filament dynamics, Yanagida and co-workers looked at the conformation of single actin monomers in the context of F-actin using fluorescence resonance energy transfer (FRET). By labeling an actin monomer with a donor and an acceptor fluorophore, the authors observed that, on the timescale of seconds, a single monomer could vary between two different conformations. Chemical cross-linking of actin, which inhibits myosin mobility but not actin binding, favored one of the two actin conformations, whereas interaction with myosin shifted the equilibrium toward the other conformation. These results suggest a molecular basis for accessory protein regulation of actin dynamics. [Letters, p. 83 ] JK

Yeast lead the way to small-molecule targets

Phenotypic screens provide a useful method for discovering small molecules that have a desired activity in vivo. However, identifying the target of the small molecule continues to be a substantial challenge. Schreiber and colleagues have now developed a new approach for characterizing the proteins and pathways targeted by small molecules. The authors grew a collection of approximately 3,900 yeast strains, each of which overexpresses a different protein, in the presence or absence of small-molecule treatment. Microarrays were then used to compare yeast strains that were enriched or depleted in response to the small molecule. As a demonstration of this approach, the authors looked at the mechanism of action of a small-molecule suppressor of rapamycin growth inhibition, LY-83583. The most resistant yeast strain overexpressed Guf1p, a protein predicted to be a GTPase located in the mitochondria. In agreement with Guf1p being a relevant target, LY-83583 inhibited Guf1p GTPase activity in vitro. [Articles, p. 103 ] JK

In This Issue written by Joanne Kotz and Terry L. Sheppard.