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This Sandbox is Reserved from 20/01/2015, through 30/04/2016 for use in the course "CHM 463" taught by Mary Karpen at the Grand Valley State University. This reservation includes Sandbox Reserved 987 through Sandbox Reserved 996.

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Contents 1 Photinus Pyralis Luciferase 1.1 Function 1.2 Mechanism 1.3 Structure 1.4 Lab Use 1.5 References

Photinus Pyralis Luciferase

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Function

Mechanism

It was believed that the chemically produced excited states stemmed from dioxetanone. This idea was proposed based on a common type of chemiluminescence which required O₂ at certain points in which dioxetanone is a precursor to the excited state. De Luca and colleagues did a study that proposed that the dioxetanone mechanism for bio- and chemiluminescence were false. Their experiment used oxygen isotopes and concluded that the oxygen atoms that the produced carbon dioxide consisted of did not stem from the consumed oxygen. This study, however, has been analyzed and several flaws have been discovered such as, incomplete chain of events and no proof of CO₂ collection from the reaction was obtainable. It was stated that the CO₂ produced was pumped directly out of the reaction. This was not possible due to the high reaction rate of CO₂and tert-butoxide ion and the stability of monoalkyl carbonates. Johnson and Shimomura determined that an oxygen atom that makes up the CO₂ does indeed stem from the O₂ consumed by the reaction in firefly bioluminescence. De Luca and colleagues reevaluated their work and their results agreed with Johnson and Shimomura. Therefore, the dioxetane-dioxetanone mechanism for firefly bioluminescence and chemiluminescence is supported.^[1]

Step one: In the photinus pyralis, the reaction begins with luciferin. Luciferase catalyzes ATP and magnesium ion to produce luciferyl AMP from luciferin.

Step 2: Luciferase then catalyzes O₂, producing light and oxyluciferin from Luciferyl AMP. ^[2][1]

Image:Screenshot 2015-02-22 22.56.09.png^[3]

Structure

Photinus pyralis luciferase is composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two domains (the N-terminal domain and the C-terminal domain) by a wide cleft. The N-terminal domain (residues 4-436) is much larger than the C-terminal domain (residues 440-544) and is formed by an antiparallet β-barrel, two β-sheets, and two α-helices.^[4] The secondary structures and motif are arranged to form a five-layered, alternating αβ tertiary structure. The C-terminal domain, on the other hand, is folded into an α+β tertiary structure.^[4]

A model for the active site of Photinus pyralis luciferase was proposed by Branchini and colleagues in 1998 and has held up to more recent data.^[5] In this model, the enzyme contains a binding pocket for ATP as well as a binding pocket for luciferin. The binding pocket for ATP is formed by the residues 316GAP318, 339GYGL342, and V362, and binds to the adenine ring.^[6] The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218.^[6] The S314-L319 loop and Q338-A348 region were found to be in different positions when substrates were bound.^[6] Since the loop blocks both of the binding pockets when in the unbound state, it makes sense that a conformational change in the loop must occur.^[6]

Lab Use

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References

1. ↑ White, E. H., Steinmetz, M. G., Miano, J. D., Wildes, P. D. and Morland, R. (1980) "Chemi- and bioluminescence of firefly luciferin", J. Am. Chem. Soc. 102(9): 3199-3208.
2. ↑ Thorne, N., Shen, M., Lea, W. A., Simeonov, A., Lovell, S., Auld, D. S. and Inglese, J. (2012) "Firefly luciferase in chemical biology: A compendium of inhibitor, mechanistic evaluation of chemotypes, and suggested use as a reporter", Chem. Biol. 19(8): 1060-1072. doi:http://dx.doi.org/10.1016%2Fj.chembiol.2012.07.015
3. ↑ Auld, D.S., Southhall, N. T., Jadhav, A., Johnson, R. L., Diller, D. J., Simeonov, A., Austin, C. P., and Inglese, J. (2008) "Characteristics of chemical libraries for luciferase inhibitory activity", J. Med. Chem. 51(8):2372-2386. doi:10.1021/jm701302v
4. ↑ ^{4.0 4.1} Conti E., Franks N.P., Brick P. (1996) "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes", Structure 4(3): 287-298. doi: 10.1016/S0969-2126(96)00033-0
5. ↑ Zako T., Ayabe K., Aburatani T., Kamiya N., Kitayama A., Ueda H., and Nagamune T. (2003) "Luminescent and substrate binding activities of firefly luciferase N-terminal domain", 1649(2): 183-189. doi: 10.1016/S1570-9639(03)00179-1
6. ↑ ^{6.0 6.1 6.2 6.3} Branchini B.R., Magyar R.A., Murtiashaw M.H., Anderson S.M., and Zimmer M. (1998) "Site-directed mutagenesis of Histidine 245 in firefly luciferase: a proposed model of the active site", Biochemistry 37(44): 15311-15319. doi: 10.1021/bi981150d.