Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA⅐MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 Å and form a clear focal point centered near the active site. We suggest that these distal anticorrelated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA⅐MD approach.anticorrelated motion ͉ correlated motion ͉ M.HhaI ͉ statistical coupling analysis T he proposal that protein dynamics contributes significantly to enzyme catalysis is intriguing (1-4) yet is supported by limited experimental evidence. Previous studies have shown that correlated and anticorrelated motions within an enzyme's active site enhance the reaction rate by various mechanisms that increase the relative amounts of reactive orientations (5). These active-site fluctuations are proposed to result from motions involving distal structural elements and interconnecting networks (1-4). This hypothesis is indirectly supported by emerging molecular dynamics (MD) (1, 2), NMR (6, 7), and hybrid approaches (8-12). The MD studies, although difficult to verify experimentally, have provided highly suggestive results relating dynamics to catalysis. Ultimately, the quantitative contribution to catalysis of various dynamic mechanisms requires direct experimental testing. We combined MD simulations and a coevolution analysis [statistical coupling analysis (SCA); ref. 13] to identify residues that are coupled by coevolution and motion.Although MD simulations reveal active-site correlated and anticorrelated motions, the identity and role of specific structural elements outside the active site in mediating such motions is difficult to assign. For example, MD cross-correlation analyses are dominated by anticorrelated motions occurring between the most distal regions of protein, often residing in distinct domains (5). Although MD simulations implicate regions of allowed motion, the identity of single amino acids that facilitate these motions is not forthcoming and hence difficult for protein engineers to test. SCA identifies the functional coupling of specific residue pairs that in many cases are distal in the three-dimensional structure. The coupling of such residues leads to their coevolution and is revealed by the statistical analysis of hundreds of related sequences; this approach recently was validated by NMR and protein engineering studies (13-16). These applications of SCA have been focused on protein-ligand interactions, and here we apply SCA toward protein dynamics and catalysis.M.HhaI is one of many S-adenosylmethionine (AdoMet)-dependent DNA-modifying enzymes found in bacteria, plants, and animals (17). These enzym...

The sulfotransferases that are active in the metabolism of xenobiotics represent a large family of enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to phenols, to primary and secondary alcohols, to several additional oxygen-containing functional groups, and to amines. Restriction of this review to the catalytic processes of phenol or aryl sulfotransferases does not really narrow the field, because these enzymes have overlapping specificity, not only for specific compounds, but also for multiple functional groups. The presentation aims to provide an overview of the wealth of phenol sulfotransferases that are available for study but concentrates on the enzymology of rat and human enzymes, particularly on the predominant phenol sulfotransferase from rat liver. The kinetics and catalytic mechanism of the rat enzyme is extensively reviewed and is compared with observations from other sulfotransferases.

A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments (outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor developed in this study was used to reveal the relative matrix:intermembrane space:outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria.Protein kinases are a large enzyme family that have been implicated in nearly every cell-based behavior, from ATP generation to unrestrained growth and division.1 These enzymes are linked by their ability to catalyze phosphoryl transfer from ATP to the hydroxyl moieties of serine, threonine, and/or tyrosine residues in proteins. A variety of factors limit protein kinasecatalyzed phosphorylation to intended protein targets: (a) the ability to phosphorylate serine/ threonine or tyrosine, but only rarely both aliphatic and aromatic residues, (b) differential expression as a function of cell type, (c) recognition of specific amino acid sequences encompassing the hydroxyl phosphoryl acceptor moiety, and (d) localization to specific intracellular sites. The cAMP-dependent protein kinase (PKA) exhibits many of these attributes as a serine/threonine-specific protein kinase with a special preference for sequences of the general form Arg-Arg-Xaa-Ser/Thr-Xaa in protein substrates.2 In addition, PKA is anchored to a variety of intracellular sites via coordination to A-Kinase Anchoring Proteins (AKAPs), and thus the biological consequences of its action are location-dependent.3 For example, mitochondrial PKA is implicated in the regulation of apoptosis and ATP synthesis.4 However, as is true for protein kinases in general, presumed intracellular PKA activity is commonly assessed in an indirect fashion: either by the mere presence of the enzyme (immunofluorescence or western blots) or by the effect of small molecule modulators, such as inhibitors, on the phosphorylation of presumed PKA protein substrates. Unfortunately, these commonly employed methods don't furnish a direct measure of kinase activity. Fluorescent sensors have been used to directly and continuously assess kinase action. 5 However, these either display a limited dynamic range or employ fluorophores with photophysical properties (short λ ex /λ em 6, small ε,7 low Φ) that are incompatible (due to interference from autofluorescence) with cells, cell lysates, or organelles. With the latter limitation in mind, we report herein the application of a quenched fluorescence strategy6 to create a kinase senso...

Protein kinases control the flow of information through cell-signaling pathways. A detailed analysis of their behavior enhances our ability to understand normal cellular states and to devise therapeutic interventions for diseases. The design and application of "Environmentally-Sensitive", "DeepQuench" and "Self-Reporting" sensor systems for studying protein kinase activity are described. These sensors allow real-time activity measurements in a continuous manner for a wide variety of kinases. As these sensors can be adapted from an in vitro screen to imaging kinase activity in living cells, they support both preliminary and later stages of drug discovery.

Assays that furnish a fluorescent readout of protein kinase activity provide a means to identify and characterize inhibitory agents, assess structure-function relationships, and correlate enzyme activity with cellular behavior. Although several protein kinase sensors have been described in the literature, their fluorescent response to phosphorylation are generally modest to moderate (1.1 -8-fold). We have developed a "deep quench" strategy that elicits a dramatic amplification of fluorescence (>60-fold) in cAMP-dependent protein kinase substrates. We describe sensor design, assay development via a combination of library synthesis and screening, characterization of the assay components, and an assessment of two inhibitory species.Protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues in protein and peptide substrates. These enzymes have received considerable attention due to the relationship between aberrant kinase activity and an assortment of human afflictions. Specific and highly sensitive protein kinase sensors furnish a means to rapidly identify inhibitors, assess protein structure/function relationships, and correlate kinase activity with cellular behavior. A large number of kinase assays have been described, however, assays with fluorescent readouts are most easily applied to both in vitro and intracellular settings. GFP-labeled protein and fluorophore-labeled peptide substrates generally deliver, upon phosphorylation, a fluorescent response that varies from 10-60% to 2-9-fold, respectively. 1 By comparison, many fluorescent sensors developed for a variety of biomolecules (e.g. proteinases 2 and the detection of specific nucleotide sequences 3 ) display enhancements of 25-fold and greater. A large dynamic range offers enhanced sensitivity, thereby furnishing a means to assess target biomolecule behavior under a variety of conditions. Unlike nearly all of the protein kinase assays reported to date, 4 the readout described in studies with proteinases 2 and molecular beacons 3 arise via relief of fluorescent quenching. We report herein an approach, devised around the latter conceptual framework ("Deep Quench"), which delivers a robust protein kinase-elicited fluorescent response.Our initial studies have focused on the strategy outlined in Scheme 1. A fluorophore-labeled kinase substrate (A) exhibits little or no fluorescence (B) in the presence of a quencher molecule. Upon phosphorylation, the peptide product (C) is sequestered by a phospho-Ser binding domain to form the complex D, which disrupts the interaction between peptidefluorophore and quencher. The latter should partially or completely restore the fluorescence of the starting peptide.Pyrene was chosen to serve as the fluorophore on an amino acid sequence (AcGRTGRRFSYPamide) recognized by the cAMP-dependent protein kinase ("PKA"). 5 We employed the phospho-Ser binding domain, 14-3-3τ, to serve as the sequestering agent since 14-3-3 domains display a high affinity for phosphoSer-containing peptides (K D < 100 nM...