SCIENCE & TECHNOLOGY
ENABLING ENZYME STUDIES Researchers unveil SOLUTIONS TO DIFFICULT PROBLEMS at mechanisms conference STU BORMAN, C&EN WASHINGTON
THREE AMBITIOUS and dynamic re-
unknown enzymes found in genome projects—to understand how enzymes make the body work and learn how useful new enzymes can be engineered. Cravatt’s strategy for ferreting out new inhibitors for multiple enzymes simultaneously is a good example “of how a strong understanding of enzyme mechanisms can serve as a starting point for studies in chemical biology,” said John P. Richard, chair of the meeting’s organizing group
COURTESY OF PATRICIA BABBITT
search concepts that promise to accelerate the study and applications of enzyme mechanisms were presented at the 22nd Enzyme Mechanisms Conference last month in St. Pete Beach, Fla. Professor of chemical physiology Benjamin F. Cravatt III of Scripps Research Institute, in La Jolla, Calif., discussed a technique for finding inhibitors for hundreds of enzymes at a time that could speed drug discovery.
Called competitive activity-based protein profiling (competitive ABPP), the technique by Cravatt and coworkers could accelerate inhibitor discovery by making it possible to identify selective inhibitors for multiple enzymes that catalyze similar reactions. Competitive ABPP is an extension of the ABPP technique that Cravatt and coworkers developed earlier. In ABPP, a fluorescenceor biotin-based tag is linked to an active-site functional group found in each member of an entire class of enzymes, such as serine hydrolases. These tags thus identify members of the enzyme class in proteomic analyses. For example, the researchers recently used a cysteine-specific reagent to identify, in proteomic mixtures, enzymes that have highly reactive cysteine residues (Nature, DOI: 10.1038/nature09472; C&EN, Nov. 29, 2010, page 8). In another ABPP study, they used reagents specific for serine hydrolase active sites to label and identify serine hydrolases in proteomic mixtures.
FAMILY TOGETHERNESS Conventional enzyme trees, such as that of the 54 homologs representing known reaction families (left; green, blue, and red) along with many unknown reactions (gray or black), display less information than similarity networks, such as that of 1,247 sequences of enzymes representing the same known reaction families as those in the tree, as well as two families (right; orange and yellow) for which function has been newly identified.
Associate professor of chemistry Kate S. Carroll of Scripps Florida described a new way to assess enzyme damage from oxidative stress, which is associated with many human diseases. And professor of biopharmaceutical sciences Patricia C. Babbitt of the University of California, San Francisco, explained computational methods to determine the function of
and a specialist on mechanisms of enzymecatalyzed reactions at the State University of New York, Buffalo. Inhibitors are useful tools for probing enzyme functions, and they are potential drug candidates. But most inhibitors are found in a slow, one-byone manner—for example, by screening a library of compounds for the ability to shut down a specific enzyme. WWW.CEN-ONLINE.ORG
28
FEBRUARY 7, 2011
“Our original idea was to use ABPP to compare normal and cancer cells, or normal and Alzheimer’s brain cells, to find aberrant enzyme activities relevant to the pathology,” Cravatt said. “The next thing you want to do is inhibit the [disease-related] enzymes” to probe their functional roles, “but few inhibitors are available. For example, there are about 250 serine hydro-
lases in humans, but until recently inhibitors had been identified for only a handful of them,” Cravatt said. To systematically identify inhibitors for whole families of enzymes, such as serine hydrolases, Cravatt and coworkers devised competitive ABPP. “The goal would be to have 250 inhibitors that inhibit selectively 250 serine hydrolases,” Cravatt said. “We’re not there yet, but we’re making pretty good progress.”
inadequate. A technique by Carroll and coworkers to identify and quantify protein oxidation could advance efforts to understand oxidative effects. Oxidative stress is toxic to cells because highly reactive oxygen species can damage proteins, lipids, DNA, and other biomolecules. For example, high cellular levels of hydrogen peroxide (H2O2) interfere with
cell function by modifying the catalytic activity, DNA-binding activity, and stability of enzymes. But peroxide also plays an important role in activating signaling pathways. So cells have to regulate peroxide levels carefully. In enzymes and other proteins, cysteine residues react with peroxide to form S-hydroxylated cysteines, also known as
IN COMPETITIVE ABPP of serine hy-
drolases, a library of potential inhibitors competes with a fluorophosphonate to react with the enzymes’ active sites (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/ pnas.1011663107). Fluorophosphonates are known to react selectively with hydroxymethyl side chains in serine hydrolase active sites. The most effective inhibitors, and the hydrolases they target, are then identified. Competitive ABPP can be extended to other enzyme families by developing other appropriate active-sitespecific reagents, Cravatt noted. Competitive ABPP thus identifies inhibitors for multiple enzymes in a single experiment. The technique also streamlines inhibitor discovery because it doesn’t require prior purification or recombinant expression of enzymes or prior identification of enzyme substrates and products—two time-consuming aspects of conventional techniques. Cravatt and coworkers hope the new inhibitors discovered through competitive ABPP will, in fact, make it easier to identify enzymes’ substrates, products, and biological roles from observations of metabolic, physiological, and proteomic changes that occur when they use the inhibitors to squelch the activity of the enzymes. The group’s newfound inhibitors could also be promising drug candidates. Serine hydrolase inhibitors discovered in the past include cholinesterase inhibitors to treat Alzheimer’s disease and dipeptidyl peptidase-4 inhibitors to treat diabetes. At Scripps Florida, Carroll and coworkers have been probing the effects of reactive oxygen species on enzymes, an area of growing research interest. Oxidative stress—unchecked imbalances of reactive oxygen species in cells—has profound effects on biological systems and is associated with diseases such as cancer and diabetes. But the effects of oxidative stress on cells are not yet completely understood, and current techniques to study them are
NEW!
True Blue Performance Exceptionally inert, Sky™ inlet liners with state-of-the-art deactivation improve trace level analysis. • Increase accuracy and precision • Lower detection limits • Use wool with confidence
WWW.CEN-ONLINE.ORG
TM
Request flyer GNFL1323 or learn more at
www.restek.com/sky
Innovative Chromatography Products Restek U.S. • 110 Benner Circle • Bellefonte, PA 16823 • 814-353-1300 • 800-356-1688 • fax: 814-353-1309 • www.restek.com Restek France • phone: +33 (0)1 60 78 32 10 • fax: +33 (0)1 60 78 70 90 • e-mail:
[email protected] Restek GmbH • phone: +49 (0)6172 2797 0 • fax: +49 (0)6172 2797 77 • e-mail:
[email protected] Restek Ireland • phone: +44 (0)2890 814576 • fax: +44 (0)2890 814576 • e-mail:
[email protected] Restek Japan • phone: +81 (3)6459 0025 • fax: +81 (3)6459 0025 • e-mail:
[email protected] Thames Restek U.K. LTD • phone: +44 (0)1494 563377 • fax: +44 (0)1494 564990 • e-mail:
[email protected] 29
FEBRUARY 7, 2011
SCIENCE & TECHNOLOGY
COURTESY OF KATE CARROLL
COU RT ESY O F BEN JAM IN CRAVAT T
sulfenic acids. These can be Babbitt and coworkers have detected by treating protein adopted a different form of repsamples with dimedone, which resentation that they believe O reacts specifically with sulfenic has advantages over traditional B: B: O acid to form an adduct that can tree models. They use a proO P P OH + be detected mass spectrometgram called Cytoscape to disF O O rically. However, determining play sequence or structure data the level of stress, which is the on enzyme family members in fraction of cysteines oxidized “similarity networks.” to sulfenic acids, requires asData in similarity networks, ACTIVITY-BASED PROBE Selective reaction between a says not only for sulfenic acid such as evolutionary distances fluorophosphonate and serine hydrolase active sites enables but also for cysteine. between family members, are marking of enzymes and competition experiments. B is a base Carroll and coworkers have consistent with similar data in that’s common to serine hydrolase active sites; the red sphere now made such determinaphylogenetic tree diagrams. is a fluorophore used for enzyme detection. tions possible by developing But similarity networks are iododimedone. This new more interactive and compact, reagent reacts with cysteine provide information not availI thiols to give the same adduct able in trees, and can easily O O O as that formed from dimedone handle many more sequences CH3 and sulfenic acids. Labeling or structures. Similarity netSH S H3C CH3 dimedone’s methyl groups works provide a better intuitive CH3 Iododimedone with deuterium provides a sense of differences in rates of O 6-dalton difference that alevolution for different families lows adducts from the two at a glance and indicate the relO O O reactions to be distinguished ative size of each reaction class CD3 by mass spectrometry (Angew. more effectively, Babbitt said. S SOH D C CD Chem. Int. Ed., DOI: 10.1002/ Researchers are also using 3 3 CD3 [D6]Dimedone anie.201007175). In principle, similarity networks—together O the method can determine with other methods such as in the relative level of thiol and silico docking, structural charsulfenic acid side chains in any acterization, and experimental RELATIVITY The reaction of iododimedone and enzyme sample. screening—to develop a genhexadeuterated dimedone with enzyme thiols and sulfenic “This capability has never eral strategy for assigning funcacid groups, respectively, yields two forms of dimedone been reported before,” Carroll tions to enzymes discovered in adducts that can be distinguished by their 6-dalton said. “It’s an extremely facile genome projects. For example, difference, thereby permitting assessment of the amount of and enabling technology.” Babbitt; John A. Gerlt of the cysteine oxidation in the enzyme. Carroll’s “group has made University of Illinois, Urbanaa very simple change in the Champaign; Matthew P. Jacobstructure of dimedone—iodination—that structure-based computational methods son of UCSF; Steven C. Almo of Albert Einchanges the reagent from a nucleophile to discover the physiological functions of stein College of Medicine; and coworkers that reacts with electrophilic sulfenic acids unknown enzymes discovered in genome recently identified a cluster of enzymes of to an electrophile that reacts with nucleoprojects and thus understand the role of unknown function in a sequence similarphilic thiols,” Richard commented. “This those enzymes in health and disease. ity network as new variants of muconate work provides an incredibly simple and lactonizing enzymes and N-succinylamino elegant solution to a difficult problem.” ASSIGNING FUNCTIONS to poorly characid racemases (Biochemistry, DOI: 10.1021/ The technique should help scientists acterized enzymes is a major problem. bi802277h; Nat. Chem. Biol., DOI: 10.1038/ prioritize proteins for further characterizaFor example, the sequences of more than nchembio.2007.11). tion and functional analysis related to oxi6,000 enzymes in the enolase superfamOther applications of sequence and dative stress. It could also be used to assess ily are known. But the functions of about structure similarity networks include disprotein modifications caused by redoxhalf those enzymes remain unknown, decovery of enzyme substrates and products modulating drugs and to look for protein spite extensive efforts by several research and of homologous relationships among markers of oxidation in various disease groups. different enzymes. They could also help states for diagnostic purposes. To organize what is known about su“guide the choice of starting scaffolds for So far, Carroll and colleagues have used perfamilies, researchers divide them into enzyme engineering,” Babbitt said. By takthe new approach only in vitro, “but the subgroups of related families that catalyze ing enzymes that nature has retooled over door is wide open for cellular studies,” different reactions but share common and over to evolve new chemistry and using she said. “We’re excited about what lies functional properties. They then use stickthem as structural templates for enzyme ahead.” like phylogenetic tree diagrams to display engineering and design, Babbitt said, “we Meanwhile, UCSF’s Babbitt and coprobable or known evolutionary relationmay be able to more successfully engineer workers have been using sequence- and ships of different enzymes in each family. new enzymatic reactions in the lab.” ■ WWW.CEN-ONLINE.ORG
30
FEBRUARY 7, 2011
