Raising Enzymes from the Dead and the Secrets They Can Tell Michael A. Marletta* Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720-1460
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e often struggle with how to extend beautifully done in vitro experiments with clear molecular explanations into the much more complicated environs of cells and complex organisms. Would it not be great if we could disable a protein of interest, an enzyme for example, and then turn it on at will? Would it not be even better if the control over activity were reversible? This is exactly what Cole and colleagues (1 ) have accomplished with an inactive mutant of the proto-oncogene, tyrosine kinase src that they chemically rescued in cells using the small molecule imidazole. This could easily just fall into the neat trick category; however, their results include two surprises. First, it appears that Src is active under basal conditions, an observation that is certain to have impact upon the signaling field. Second, they turn up new substrates for the kinase including CrkL (chicken tumor virus no. 10 [CT10] regulator of kinase). As the authors point out “CrkL is a particularly intriguing Src target because of its well-established role in cytoskeletal signaling and its known Src connections”. Src is a tyrosine kinase and the first of that family to be described. The story begins with Peyton Rous and what he endured to convince the community that what was later identified and named Rous sarcoma virus was involved in tumor formation. In time it was shown that v-src is an oncogene and essential to the transforming properties of the virus. It is an understatement to say that finding the cellular homologue c-src was a major step forward along with the later identificawww.acschemicalbiolog y.o rg
tion of it as a proto-oncogene. Many key discoveries followed and continue up to the present. The Nobel Prizes to Rous, Varmus, and Bishop certainly add to the luster of the great discoveries. Steve Martin has written two very nice accounts of how we have gotten to this point (2, 3 ). Despite all that has been uncovered, Src proteins are tyrosine kinases, how mutations in src can lead to transformed cells, and much more, functional questions remain unanswered. The findings by Cole and colleagues provide new insight into the long-standing questions. Site-directed mutagenesis has been a powerful tool, particularly in the probing enzyme mechanism (4 ). Mutagenesis to change enzyme specificity represents an important early contribution showing that enzyme engineering was indeed possible. Though many examples exist, the proteases remain among the most instructive (5 ). The replacement of key protein sidechains that lead to little or no activity and the regaining of activity by building that missing, chemically important residue into the substrate, termed chemical complementation, was an important forerunner to chemical rescue (6 ). This led the way to chemical rescue with what has also been termed substrate-assisted catalysis, creation of an inactive site directed mutant of a residue involved in catalysis and the rescue of activity by providing a substrate analog the contains chemical functionality that is missing from the mutant residue (7 ). In this work, Carter and Wells removed the catalytic histidine from B. subtilis subtilisin and replaced it with alanine. The enzyme
A B S T R A C T The complexity of partners and reaction sequence has made the deciphering eukaryotic signaling pathways particularly difficult. Various approaches have yielded important results. A recent paper reports on an advance that uses chemical rescue of inactive mutant of Src. The rescue was done in cells, and the results obtained showing new aspects of Src function point the way toward a general use of this chemical tool in the sorting out of complex biological processes.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online March 17, 2006 10.1021/cb600110g CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. Chemical rescue of Src. a) wild type c-Src showing the active site D386 and R388 (PDB id entry 2PTK); b) model of R388A made by removal of R388 except for the b-carbon. This mutation renders the enzyme inactive; and c) model shown in b) with imidazole placed in the site occupied by R388 in the wild type protein. The addition of imidazole resurrects catalytic activity. The figures were made by Douglas Mitchell at University of California-Berkeley using PyMol (http://www.pymol.org).
was severely wounded (kcat/Km was down a million-fold); however a substrate with a histidine in the P2 position complemented the missing catalytic histidine and significant activity was restored. The first true chemical rescue was carried out by Toney and Kirsch on aspartate aminotransferase, a pyridoxal phosphate dependent enzyme (8 ). In this remarkable paper, they removed a critical lysine residue that acts a general base in the essential proton transfer steps in the reaction and replaced it with alanine. The resulting mutant was dead but could be rescued by the addition of exogenous amines. Toney and Kirsch ultimately used 11 different amines and were able to do the first true Brønsted analysis of proton transfer reactions in enzymes (8 ). Now, Cole and colleagues (1 ) used imidazole to rescue an inactive Src mutant, R388A. Arginine 388 is conserved in the Src family and participates in hydrogen bonds with the substrate tyrosine hydroxyl group and an aspartate active site residue, again conserved in the Src family. The inactive mutant is then rescued by imidazole playing the part of the mutated arginine (1) Imidazole rescue has received attention before, and these past studies have shown the utility of using it to decipher complex reactions and in heme cofactor binding. For example, Ortiz de Montellano and colleagues (9 ) restored catalytic activity to an inactive mutant (H25A) of heme oxygenase with imidazole. In this case, the added imidazole replaces the proximal His ligand to the heme when bound as a substrate. Goodin and co-workers carried out similar studies with cytochrome c peroxidase 74
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(10 ). Others have rescued heme cofactor binding with imidazole where the histidine mutation has taken away the proximal ligand to the heme (11, 12 ). Expression without imidazole leads to apoprotein, and so these latter two studies also strongly suggest that His–iron coordination is an early step in cofactor incorporation, perhaps early along the folding path. The choice of imidazole to replace arginine by Cole and colleagues (1 ) circumvents the transport and toxicity issues of guanidines. Imidazole itself is not without toxicity, but they were able to carry out the studies without cellular toxicity as a complicating problem. It is somewhat surprising that 30–50% of the wild-type activity is regained with imidazole when attempting to replace arginine, but it works. With the cellular experiments, Cole and colleagues (1 ) have taken rescue to a new level and they chose a target where there are plenty of important questions to answer. Despite years of intense study, the cellular functions of Src remain elusive. The ability to control the enzyme activity in cells without laborious conditional knockouts should open new doors, and it has. Addition of imidazole in the absence of growth factor stimulation still led to phosphorylation of tyrosine, so c-Src is basally active. Future experiments that combine stimulation with control of activity will be very useful in sorting out complex signaling pathways. Perhaps most importantly, the authors identify new substrates (CrkL, lamin A/C, and procollagan). Findings like these will prove invaluable to a complete understanding of the complex signaling pathways regulated by proteins like Src.
Chemical rescue is clearly complementary to the use of inhibitors and has some advantages. The paper from Cole and colleagues (1 ) illustrates the power of the method and points the way toward future discoveries. REFERENCES 1. Qiao, Y., Molina, H., Pandey, A., Zhang, J., and Cole, P. A. (2006) Chemical rescue of a mutant enzyme in living cells, Science 311, 1293–1297. 2. Martin, G. S. (2001) The hunting of the Src, Nat. Rev. Mol. Cell Biol. 2, 467–475. 3. Martin, G. S. (2004) The road to Src, Oncogene 23, 7910–7917. 4. Peracchi, A. (2001) Enzyme catalysis: removing chemically “essential” residues by site-directed mutagenesis, Trends Biochem. Sci. 26, 497–503. 5. Craik, C. S., Largman, C., Fletcher, T., Roczniak, S., Barr, P. J., Fletterick, R., and Rutter, W. J. (1985) Redesigning trypsin: alteration of substrate specificity, Science 228, 291–297. 6. Hwang, Y. W., and Miller, D. L. (1987) A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein, J. Biol. Chem. 262, 13081–13085. 7. Carter, P., and Wells, J. A. (1987) Engineering enzyme specificity by “substrate-assisted catalysis”, Science 237, 394–399. 8. Toney, M. D., and Kirsch, J. F. (1989) Direct Bronsted analysis of the restoration of activity to a mutant enzyme by exogenous amines, Science 243, 1485–1488. 9. Wilks, A., Sun, J., Loehr, T. M., and Ortiz de Montellano, P. R. (1995) Heme oxygenase His25Ala mutant: replacement of the proximal histidine iron ligand by exogenous bases restores catalytic activity, J. Am. Chem. Soc. 117, 2925–2926. 10. McRee, D. E., Jensen, G. M., Fitzgerald, M. M., Siegel, H. A., and Goodin, D. B. (1994) Construction of a bisaquo heme enzyme and binding by exogenous ligands, Proc. Natl. Acad. Sci. U.S.A. 91, 12847–12851. 11. Barrick, D. (1994) Replacement of the proximal ligand of sperm whale myoglobin with free imidazole in the mutant His-93-->Gly, Biochemistry 33, 6546–6554. 12. Zhao, Y., Schelvis, J. P., Babcock, G. T., and Marletta, M. A. (1998) Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand, Biochemistry 37, 4502–4509.
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