Observation of unstable species in enzyme-catalyzed transformations using protein crystallography

Current Opinion in Chemical Biology

Volumn 4, Issue 1, 2000, Pages 89-94

  Petsko, Gregory A ^a   Ringe, Dagmar ^a  

^a BRANDEIS UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ENZYME; MUTANT PROTEIN;

CATALYSIS; CATALYST; CHEMICAL MUTAGENESIS; CRYSTAL; CRYSTALLOGRAPHY; ENZYME CHEMISTRY; LOW TEMPERATURE PROCEDURES; PROTEIN STRUCTURE; REVIEW; STRUCTURE ANALYSIS;

EID: 0033961309     PISSN: 13675931     EISSN: None     Source Type: Journal    
DOI: 10.1016/S1367-5931(99)00057-5     Document Type: Review

References (41)

1. Ringe D. Can Laue catch Maxwell? Philos Trans R Soc Lond A. 340:1992;243-252.
2. Douzou P., Petsko G.A. Proteins at work: 'stop-action' pictures at subzero temperatures. Adv Protein Chem. 36:1984;245-361.
3. Moffat K. Laue diffraction. Carter C., Sweet R.M. Methods in Enzymology. 1997;433-447 Academic Press, San Diego.
4. Stoddard B.L., Dean A., Bash P.A. Combining Laue diffraction and molecular dynamics to study enzyme intermediates. Nat Struct Biol. 3:1996;590-595.
5. Neutze R., Hajdu J. Femtosecond time resolution in X-ray diffraction experiments. Proc Natl Acad Sci USA. 94:1997;5651-5655.
6. Clifton I.J., Duke E.M.H., Wakatsuki S., Ren Z. Evaluation of Laue diffraction patterns. Carter C., Sweet R.M. Methods in Enzymology. 1997;448-467 Academic Press, San Diego.
7. Scott W.G., Murray J.B., Arnold J.R.P., Stoddard B.L., Klug A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science. 274:1996;2065-2069.
8. Ridder I.S., Rozeboom H.J., Kalk K.H., Dijkstra B.W. Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase. J Biol Chem. 274:1999;30672-30678. Dijkstra and co-workers continue their beautiful series of studies of the mechanism of action of L-2-haloacid dehalogenase with this use of low pH to trap a covalent ester-enzyme intermediate of the nucleophile, Asp8. This structure leads to an explanation of the stereospecificity and limited substrate specificity of the enzyme.
9. Demmel F., Doster W., Petry W., Schulte A. Vibrational frequency shifts as a probe of hydrogen bonds: thermal expansion and glass transition of myoglobin in mixed solvents. Eur Biophys J. 26:1997;327-335.
10. Ding X., Rasmussen B.F., Petsko G.A., Ringe D. Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase. Biochemistry. 33:1994;9285-9293.
11. Rasmussen B.F., Stock A.M., Ringe D., Petsko G.A. Crystalline ribonuclease A loses function below the dynamical transition at 220K. Nature. 357:1992;423-424.
12. Hope H. Crystallography of biological macromolecules at ultra-low temperature. Annu Rev Biophys Biophys Chem. 19:1990;107-126.
13. Burzlaff N.I., Rutledge P.J., Clifton I.J., Hensgens C.M., Pickford M., Adlington R.M., Roach P.L., Baldwin J.E. The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. Nature. 401:1999;721-724. Flash-freezing of the enzyme-substrate complex and of a complex with a substrate analog produces structures of two key intermediates in the reaction cycle of the enzyme that synthesizes the β-lactam ring of the penicillins. Particularly noteworthy is the use of a high pressure of molecular oxygen gas as the trigger for the reaction.
14. Edman K., Nollert P., Royant A., Belrhali H., Pebay-Peyroula E., Hajdu J., Neutze R., Landau E.M. High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature. 401:1999;822-826. After years of effort, true three-dimensional crystals of bacteriorhodopsin were obtained recently and the structure of the protein by conventional crystallographic methods confirmed Henderson and Unwin's pioneering structure obtained with electron diffraction on two-dimensional crystals. Now the first high-resolution structure of a photocycle intermediate has been obtained by constant illumination of frozen crystals.
15. Petsko G.A. Art is long and time is fleeting. Philos Trans R Soc Lond A. 340:1992;323-331.
16. Kack H., Gibson K.J., Lindqvist Y., Schneider G. Snapshot of a phosphorylated substrate intermediate by kinetic crystallography. Proc Natl Acad Sci USA. 95:1998;5495-5500. Another example of the power and versatility of flash-freezing as a means of trapping enzyme intermediates in protein crystals. Because the reaction has a large temperature coefficient the addition of substrate could be used as a trigger. A particularly nice feature of this study is that the same intermediate was observed by two different procedures. Such controls are not as common as they should be in time-resolved crystallography.
17. Choi K.H., Mazurkie A.S., Morris A.J., Utheza D., Tolan D.R., Allen K.N. Structure of a fructose-1,6-bis(phosphate) aldolase liganded to its natural substrate in a cleavage-defective mutant at 2.3 Å Biochemistry. 38:1999;12655-12664. In this study, mutagenesis is used to remove a crucial catalytic group, thereby arresting the reaction at a stage in which a single species accumulates to high occupancy and can be observed in the crystal. The structure implies that a large movement in the position of the substrate occurs upon formation of the covalent Schiff base intermediate.
18. Pannifer A.D., Flint A.J., Tonks N.K., Barford D. Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by X-ray crystallography. J Biol Chem. 273:1998;10454-10462. An extremely clever and general approach to the trapping of intermediates in protein phosphatase reactions - the removal of a glutamine sidechain that positions and polarizes the water nucleophile - is here used to trap the covalent cysteinyl-phosphate intermediate for high-resolution structure determination. A conformational change is observed in which a flexible loop folds down over the active site during catalysis.
19. Pai E.F., Krengel U., Petsko G.A., Goody R.S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 Å resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9:1990;2351-2359.
20. Skarzynski T., Kim D.H., Lees W.J., Walsh C.T., Duncan K. Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA. Biochemistry. 37:1998;2572-2577.
21. Rhee S., Miles E.W., Mozzarelli A., Davies D.R. Cryocrystallography and microspectrophotometry of a mutant (alpha DGON) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60. Biochemistry. 37:1998;10653-10659.
22. Reyes C.L., Sage C.R., Rutenber E.E., Nissen R.M., Finer-Moore J.S., Stroud R.J. Inactivity of N229A thymidylate synthase due to water-mediated effects: isolating a late stage in methyl transfer. J Mol Biol. 284:1998;699-712.
23. Zhang Z., Komives E.A., Sugio S., Blacklow S.C., Narayana N., Xuong N.H., Stock A.M., Petsko G.A., Ringe D. The role of water in the catalytic efficiency of triosephosphate isomerase. Biochemistry. 38:1999;4389-4397.
24. Fitzpatrick P.A., Steinmetz A.C., Ringe D., Klibanov A.M. Enzyme crystal structure in a neat organic solvent. Proc Natl Acad Sci USA. 90:1993;8653-8657.
25. Farber G.K. Crystallographic analysis of solvent-trapped intermediates of chymotrypsin. Methods Enzymol. 308:1999;201-215.
26. Yennawar N.H., Yennawar H.P., Farber G.K. X-ray crystal structure of gamma-chymotrypsin in hexane. Biochemistry. 33:1994;7326-7336.
27. Schmitke J.L., Stern L.J., Klibanov A.M. Comparison of X-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water. Proc Natl Acad Sci USA. 95:1998;12918-12923. Any hydrolytic reaction can be arrested by the simple expedient of carrying out the reaction in an anhydrous solvent. It is easier than one might think to stabilize many protein crystals in such solvents [24]. This paper shows how the technique can be exploited in the study of a serine protease of great commercial importance.
28. Alber T.C., Davenport R.C. Jr., Giammona D.A., Lolis E., Petsko G.A., Ringe D. Crystallography and site-directed mutagenesis of yeast triosephosphate isomerase: what can we learn about catalysis from a 'simple' enzyme? Cold Spring Harb Symp Quant Biol. 52:1987;603-613.
29. Lavie A., Allen K.N., Petsko G.A., Ringe D. X-ray crystallographic structures of D-xylose isomerase-substrate complexes position the substrate and provide evidence for metal movement during catalysis. Biochemistry. 33:1994;5469-5480.
30. Teplyakov A., Obmolova G., Badet-Denisot M.A., Badet B. The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate isomerase. Protein Sci. 8:1999;596-602. The simplest method to 'trap' an intermediate can be used with any isomerase, racemase, mutase or epimerase: simply soak the substrate (or product) into the crystals and allow the single-substrate/single-product reaction to settle to equilibrium [28,29]. In this study, exposure of an isomerase to product is used to visualize the enzyme-product complex without the need for freeze-trapping or other special techniques.
31. Farber G.K., Glasfeld A., Tiraby G., Ringe D., Petsko G.A. Crystallo-graphic studies of the mechanism of xylose isomerase. Biochemistry. 28:1989;7289-7297.
32. Petsko G.A. Flow cell construction and use. Methods Enzymol. 114:1985;141-146.
33. Wyckoff H.W., Doscher M., Tsernoglou D., Inagami T., Johnson L.N., Hardman K.D., Allewell N.M., Kelly D.M., Richards F.M. Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S. J Mol Biol. 27:1967;563-578.
34. Schneider T.R., Gerhardt E., Lee M., Liang P.H., Anderson K.S., Schlichting I. Loop closure and intersubunit communication in tryptophan synthase. Biochemistry. 37:1998;5394-5406. A wonderful example of the use of a technique that should have widespread applicability. A flow cell is used to establish a pseduo-steady-state in the enzyme crystal, allowing direct observation at ordinary temperatures of the species prior to the rate-determining transition state.
35. Xiao B., Singh S.P., Nanduri B., Awasthi Y.C., Zimniak P., Ji X. Crystal structure of a murine glutathione S-transferase in complex with a glutathione conjugate of 4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence of signaling across the dimer interface. Biochemistry. 38:1999;11887-11894.
36. van Asselt E.J., Thunnissen A.M., Dijkstra B.W. High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment. J Mol Biol. 291:1999;877-898.
37. White A., Tull D., Johns K., Withers S.G., Rose D.R. Crystallographic observation of a covalent catalytic intermediate in a beta-glycosidase. Nat Struct Biol. 3:1996;149-154.
38. Sulzenbacher G., Mackenzie L.F., Wilson K.S., Withers S.G., Dupont C., Davies G.J. The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 Å resolution. Biochemistry. 38:1999;4826-4833. A fluorinated substrate analog generates an intermediate in the reaction that is stable enough for crystallographic analysis. This study is especially noteworthy for the extraordinarily high resolution, which permits the geometry of the intermediate to be analyzed in detail.
39. Li Y.F., Hata Y., Fuji T., Kurihara T., Esaki N. X-ray structure of a reaction intermediate of L-2-haloacid dehalogenase with L-2-chloropropionamide. J Biochem (Tokyo). 124:1998;20-22.
40. Mattos C., Rasmussen B., Ding X., Petsko G.A., Ringe D. Analogous inhibitors of elastase do not always bind analogously. Nat Struct Biol. 1:1994;55-58.
41. Kern D., Volkman B.F., Luginbuhl P., Nohaile M.J., Kustu S., Wemmer D.E. Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature. 402:1999;894-898.