Proteomes enzyme classes

Adam GC, Sorensen EJ, Cravatt BF (2002) Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat Biotechnol 20 805-809... 

Protein kinases catalyze the transfer of the 7-phosphate moiety of ATP to the corresponding protein substrate. More than 30% of all eukaryotic proteins are phosphorylated, with the majority of the modifications occurring on serine or threonine residues 89 Kinases constitute the largest enzyme class in eukaryotic proteomes with more than 500 members encoded in the human genome90 and play a central role in most signal transduction pathways... 

An activity-based probe meeting these requirements could, in principle, enable the comparative measurement and molecular identification of all the active members of a given enzyme class present in one or more proteomes. Importantly, these enzyme activity profiles can be read out in a variety of formats including gels [20,25], microarrays [26], liquid chromatography-mass spectrometry (LC-MS) [27], and capillary electrophoresis [28] (Fig. 7.3-3). 

The identification of enzymes selectively expressed by tumor cells and tissues may provide a rich source of new biomarkers and targets for the diagnosis and treatment of cancer. In one such effort, the activity, subcellular distribution, and glycosylation state of members from the SH superfamily of enzymes was quantitatively profiled across a panel of human cancer cell lines [20]. The SHs represent one of the largest and most diverse enzyme classes in higher eukaryotic proteomes, consisting of proteases, lipases, esterases,... 

The benefit of addressing the proteome at the level of distinct enzyme classes, as well as the versatility of ABPP reagents, is highlighted in a third example of comparative ABPP profiling. In this study, carried out by Greenbaum and colleagues, activity-based probes were applied to characterize the functional role of the papain subclass of cysteine proteases in the Plasmodium falciparum life cycle [47]. While cysteine proteases are known to be essential for the... 

The a-chloroacetamide group has features that are beneficial for undirected ABPP. Its small size does not bias binding elements towards a specific class of enzyme, and it possesses reactivity towards a broad variety of nucleophilic amino acid residues. A library of a-chloroacetamide-based probes were synthesized by Cravatt s group. The binding element in these probes was a dipeptide that was varied with small, large, hydrophobic, and charged side chains, and a biotin or rhodamine tag was appended as a reporter tag. Upon screening of eukaryotic proteomes with this library, many enzymes previously unaddressed by directed ABPP probes were uncovered. These included fatty acid synthase, hydro-xypyruvate reductase, malic enzyme, and the nitrilase superfamily [163, 164]. In contrast to the sulfonate esters, a-chloroacetamides react preferentially with cysteine residues in the proteome. 

In the recent literature, many examples of A/BPs containing benzophenones can be found. A first example concerns the study of HDACs. These enzymes catalyze the hydrolysis of acetylated lysine amine side chains in histones and are thus involved in the regulation of gene expression. There are approximately 20 human HDACs, which are divided into three classes (I, II, and III). Class I and II HDACs are zinc-dependent metallohydrolases that do not form a covalent bond with their substrates during their catalytic process, which is similar to MMPs. It has been found that hydroxamate 65 (SAHA, see Fig. 5) is a potent reversible inhibitor of class I and II HDACs. In 2007, Cravatt and coworkers reported the transformation of SAHA into an A/BP by installment of a benzophenone and an alkyne moiety, which resulted in SAHA-BPyne (66) [73]. They showed that the probe can be used for the covalent modification and enrichment of several class I and class II HDACs from complex proteomes in an activity-dependent manner. In addition, they identified several HDAC-associated proteins, possibly arising from the tight interaction with HDACs. Also, the probe was used to measure differences in HDAC content in human disease models. Later they reported the construction of a library of related probes and studied the differences in HDAC labeling [74], Their most... 

---