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Sheila Macintyre Biochemistry Pet Enzyme Project Enyzmes Are Biomolecules Typically Proteins. Enzymes Are Specific to the Subs

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Sheila Macintyre Biochemistry Pet Enzyme Project Enyzmes Are Biomolecules Typically Proteins. Enzymes Are Specific to the Subs Sheila MacIntyre Biochemistry Pet Enzyme Project Enyzmes are biomolecules typically proteins. Enzymes are specific to the substrate they will act on and will convert the molecule to a different product. All biological processes require enzymes that will determine the metabolic pathways that occur in that cell. Enzymes catalyze chemical reactions by lowering the activation energy needed in the reaction. Bioluminescence is an enzyme catalyzed reaction and each luciferin-luciferase is specific to the species in the light emitting reaction. Fireflies communicate to each other by a series of flashing. Luciferase is a 62 KDa enzyme that catalyzes the reaction between luciferin, Mg-ATP and molecular oxygen to produce an electronically excited oxyluciferin an adenylate intermediate (Conti et al. Luciferin is in the classification of oxidoreductase enzyme. In the first step, in the reaction is the formation of an acid anhydride between the carboxylic group and AMP, with the release of diphosphate. Visible light is emitted during the release of a photon from the excited state of oxyluciferin to its ground state. The luciferase reaction is a “cold” reaction, which all of the chemical energy is converted to light energy with virtually no heat produced. Below is the reaction mechanism proposed for the firefly luciferin: Luciferin + ATP luciferyl adenylate + PPi Luciferyl adenylate + O2 oxyluciferin + AMP + CO 2 + hv Figure 1: Luciferin-luciferase reaction mechanism for firefly luciferase. N S N S Mg2+ luciferase S N HO O S N HO O AMP O O O O O P OH O2 O HO N S * + CO2 + AMP + hv S N O O Primary structure of an enzyme dictates its function. The primary structure of an enzyme is Luciferase is a polypeptide containing 550 amino acid residuals. Luciferase evolved from the metabolic pathway to alleviate oxidative stress in the organisms. The light reaction occurs in a photocyte, which contains many peroxisomes and mitochondria. Figure 2. Primary sequence of firefly luciferase (1). >1LCI:A|PDBID|CHAIN|SEQUENCE MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYA EYFEMSVRLAEAMKRYGLNTNHRIVV CSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQK ILNVQKKLPIIQKIIIMDSKTDYQG FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTAC VRFSHARDPIFGNQIIPDTAILSV VPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFA KSTLIDKYDLSNLHEIASGGAPLS KEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVD LDTGKTLGVNQRGELCVRGPMIMSG YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAEL ESILLQHPNIFDAGVAGLPDDDAGEL PAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDAR KIREILIKAKKGGKSKL The secondary structure of luciferase contains 31% alpha helices with 173 residues and 22% beta sheet containing 34 strands with 124 residues. The subunit 1lci001 (red) contains 153 residues, subunit 2 (purple) 142 residues, subunit 3(blue) contains 77 residues and subunit 4 (yellow) contains 99 residues. Figure 3 Secondary Structure of Luciferase (1). The Kyte-Doolittle hydropathy plot allows the researcher to observe a more detailed structure of the protein. The number on the y-axis describes the polar or non-polar nature of each amino acid in the protein chain. Negative values indicate the hydrophilic amino acids and the positive numbers indicate the hydrophobic groups. The luciferin molecule there is an even distribution of hydrophobic and hydrophilic groups. The hydrophobic groups are important to lower contact with the solvent this will decrease the energy level and therefore change the wavelength of the light (red shift). This has been very useful in vitro experiments where higher wavelengths are required for bioluminescent imaging. Genes manipulated with solvents give scientists a broader range of colors of to use in imaging. There are two major subunits in the luciferase molecule. The N5’ begins with the blue ribbon and the C 3’ end is in red. The smaller subunit is in the red to orange region and the active site is where the red meets the blue ribbons. Lysine 443 and Lys 529 are located on opposite sides of the C terminal end and are extremely important in the oxidation reaction of the luciferin molecule (2). Figure 4 Tertiary Structure of luciferase. Ligands Figure 5 Tertiary Structure of luciferase with the Mg-AMP complex in the active site. Figure 6 Binding sites of firefly luciferase (2). The ligands present in the luciferase structure are the Cl- ion and 5-O-[N- (Dehydroluciferal)-Sulfamoyl] Adenosine. They are located in the active site and this gives the energy needed to drive the oxidation reaction to produce an excited molecule. Reaction Mechanism This reaction mechanism is important to my thesis for it explains how the Keto-enolate tautomerism will affect the color of the firefly’s lantern. The reaction also shows the conformation change in the oxyluciferin will also cause a color change. Figure 7Branchini firefly luciferase reaction mechanism, showing the keto-enol tautomerism (2). Catalytic Parameters Catalytic residues are indicated in red . Chain: A 10 20 30 40 50 60 70 80 90 100 | | | | | | | | | | AKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPV LGALFIGVAVA 110 120 130 140 150 160 170 180 190 | | | | | | | | | PANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTI ALIMNSLPKGV 210 220 230 240 250 260 270 280 290 300 | | | | | | | | | | ALPHRTACV RFSHARDPIFGNQIIPDTAILSVVPFH HGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAK STLIDKYDLSN 310 320 330 340 350 360 370 380 390 400 410 | | | | | | | | | | | LHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGL TETTSAILITPEGPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG YVNNPEATNAL 420 430 450 460 470 480 490 500 510 | | | | | | | | | IDKDGWLHSGDIAYWDEDEHFFIVLI KYKGY QVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQ VTTAKKLRGGV 520 530 540 | | | VFVDEVP KLDARKIREILIKAKK Catalytic Parameters The Michaelis Constant for firefly luciferase is Km= 0.00025 at a pH 8.6. The Ki = 0.9. Turnover rate of the reaction is 1.6 1/s. The pH range 8.6 – 6.0 at an optimal temperature range of 37-45 degrees Celsius. Enzyme Regulation The firefly luciferase enzyme is regulated by Mg-ATP providing the energy for the reaction. The Mg-ATP acts to transfer the O2 to bond in the peroxy-intermediate that provides the reaction with the energy to move the electron into an excited state. Temperature, pH, and solvent effects will also determine if the reaction will move forward. References 1. Protein Data BanK website, Retrieved August 4, 2008. <http://www.rcsb.org/pdb/explore.do?structureId=1LCI> 2. Branchini, B.R., Southworth, T.L., Murtiashaw, M.H., WilKinson, S.R., KhattaK, N.F., Rosenberg, J.C., Zimmer, M. 2005. Biochemistry, 44 : 1385-1393. 3. Protepedia: Life in 3D. Retrieved from website August 7, 2008. <http://proteopedia.org/wiki/index.php/1ry2 Sheila MacIntyre PET Enzyme Web quest Audience: This activity is for advanced level high school students with a bacKground in chemistry and biology or biochemistry students that have previous Knowledge about the functions of primary, secondary and tertiary enzyme structures. This activity will taKe two 45- minute class periods. Objective : • Identify and research the different enzyme structures of luciferin in various bioluminescent organisms using the internet. • Navigate the Protein BanK Web site • Create a power point presentation on your research and present findings to your peers. Introduction: There are dozens of luciferin-luciferase molecules that are used by organisms to produce light. In groups of three, you will create a power point slide show on the luciferin molecule of your choice. Here are a few good websites to search for your molecule and bacKground information. http://www.lifesci.ucsb.edu/~biolum/ http://www.mbari.org/topics/biology/bio-midwater.htm#biolum http://www.biology.pl/baKterie_sw/index_en.html http://www.herper.com/Bioluminescence.html http://www.seasKy.org/deep-sea/biolumiscence.html http://at-sea.org/missions/deepscope3/flourescence.html Presentation requirements 1. Include bacKground information on the organisms that use this luciferin-luciferase complex. 2. Using the Protein Data BanK website http://www.rcsb.org/pdb/home/home.do complete a search for your luciferase. 3. You may use the tutorial for Green Fluorescent Protein as an example. http://www.rcsb.org/pdb/education_discussion/educational_resources/bioinformatics_ tutorial_gfp.pdf 4. Include in the presentation slides showing the reaction mechanism for your luciferase, the Primary Sequence, Secondary sequence, tertiary sequence, catalytic parameters of your luciferase. 5. Show the hydrophobic and hydrophilic portions of your molecule. 6. If there are any ligands, accessory pigments or co factors involved in the mechanism include them in your presentation. 7. Conclude with an application or current research that is involved with your luciferase enzyme. .
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