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Many enzymes use ATP in signal-transducing processes or as an energy source. New fluorogenic ATP analogues signal ATP consumption by ubiquitin-like protein-activating enzymes in real time. Thus the inhibition and stimulation of these ATP-processing enzymes can be studied without auxiliary enzymes and reagents. beta-Lapachone was identified as an inhibitor of the ubiquitin-activating enzyme UBA1 (see scheme; A=acceptor, D=donor).

The X-linked dominant trait focal dermal hypoplasia (FDH, Goltz syndrome) is a developmental defect with focal distribution of affected tissues due to a block of Wnt signal transmission from cells carrying a detrimental PORCN mutation on an active X-chromosome. Molecular characterization of 24 unrelated patients from different ethnic backgrounds revealed 23 different mutations of the PORCN gene in Xp11.23. Three were microdeletions eliminating PORCN and encompassing neighboring genes such as EBP, the gene associated with Conradi-Hunermann-Happle syndrome (CDPX2). 12/24 patients carried nonsense mutations resulting in loss of function. In one case a canonical splice acceptor site was mutated, and 8 missense mutations exchanged highly conserved amino acids. FDH patients overcome the consequences of potentially lethal X-chromosomal mutations by extreme skewing of X-chromosome inactivation in females, enabling transmission of the trait in families, or by postzygotic mosaicism both in male and female individuals. Molecular characterization of the PORCN mutations in cases diagnosed as Goltz syndrome is particularly relevant for genetic counseling of patients and their families since no functional diagnostic test is available and carriers of the mutation might otherwise be overlooked due to considerable phenotypic variability associated with the mosaic status.

The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different ATPases suitable tools are required. Here, we present the synthesis of six new ATP-based ATPase probes modified at three different positions of the nucleobase and the ribose, respectively. Subsequently, we studied the ATPases focal adhesion kinase FAK, the ubiquitin-activating protein UBA1 and the kinesin Eg5 as examples for ATPases that process ATP by different mechanisms. We find that for each of these enzymes at least one position in ATP can be modified without loss of acceptance by the enzyme. However, the positions at which modifications are tolerated significantly differ between the studied enzymes allowing fingerprints to be drawn for reactivity. The introduced ATP analogues may form the basis for the design of tailored probes with increased affinity and specificity for a specific ATPase of interest.