Sandbox Reserved 993: Difference between revisions

''Photinus pyralis'' luciferase a monomeric enzyme composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two <scene name='69/691535/Overall_structure_domains/1'>domains</scene> (the N-terminal domain and the C-terminal domain) by a wide cleft. Although not shown in the model, the domains (N-terminal in green and C-terminal in blue) are connected by a flexible loop structure. 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.<ref name=Conti1996>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</ref> 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.<ref name=Conti1996 /> Currently, it is thought that the active site is located at the surfaces where the domains meet and that a conformation change occurs after the substrates are bound in which the domains come together and enclose the substrates. <ref name=Conti1996><ref name=Marques2009>Marques S.M. and Esteves da Silva J.C.G. (2009) "Firefly bioluminescence: mechanistic approach of luciferase catalyzed reactions", IUBMB Life 61(1): 6-17. doi: 10.1002/iub.134<ref/> This enclosement creates a hydrophobic environment which prevents light production from being quenched by water. <ref name=Conti1996><ref name=Bedford2012>Bedford R., LePage D., Hoffman R., Kennedy S., Gutschenritter T., Bull L., Sujijantarat N., DiCesare J.C., and Sheaff R.J. (2012) "Luciferase inhibition by a novel naphthoquinone", J. Photochem. Photobiol., B 107: 55-64. doi: 10.1016/j.jphotobiol.2011.11.008<ref/>

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.<ref name=Zako2003>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</ref> 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.<ref name=Branchini1998>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.</ref> The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218.<ref name=Branchini1998 /> A model of the active site with a bound luciferase inhibitor (PTC128) is shown <scene name='69/691535/Active_site_structure/2'>here</scene> (blue=ATP binding pocket, purple=luciferin binding pocket, and green=residues shared by binding pockets). The S314-L319 loop and Q338-A348 region were found to be in different positions when substrates were bound.<ref name=Branchini1998 /> 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.<ref name=Branchini1998 />