Griffith: Nitrate Reductase
Eukaryotic nitrate reductase is a cytoplasmic enzyme that functions as either a homodimer or a homotetramer of identical subunits (100 kD each) and catalyzes the first step in the reduction of nitrate to ammonium for cellular nitrogen assimilation. Each monomer is comprised of eight functional sequence regions and incorporates one of each of flavin adenine dinucleotide (FAD), heme-Fe (cytochrome-b) and Mo-molybdopterin (Mo-MPT). Eukaryotic nitrate reductase can utilize NADH and/or NADPH as an electron donor. The mechanism of nitrate reductase incorporates a redox reaction that relies on an internal electron transport chain between two mutually exclusive active sites. FAD is reduced by NAD(P)H in the first active site and passes electrons through the heme-Fe center to the second active site where the Mo-MPT is reduced. Reduced MoIV transfers electrons to nitrate to produce nitrite and hydroxide. The electrons flow according to the redox potential of each cofactor in the homodimer form of the enzyme, from FAD with NAD+ bound (-210 mV) to cytochrome-b (-160 mV) to Mo (0 mV). The large difference in redox potential indicates the flow of electrons is thermodynamically favorable and that most of the electrons will be found at the Mo-MPT active site at equilibrium. This molecular model shows the Mo-MPT active site within the nitrate reductase enzyme of the thermotolerant yeast, Pichia Angusta, and highlights the position of the required molybdenum cofactor.
|Jennifer Hiras: HMG-CoA Reductase
HMG-CoA reductase is the rate-limiting enzyme for cholesterol synthesis where it catalyzes the production of mevalonate from HMG-CoA. This makes HMG-CoA reductase is a primary target in clinical studies to reduce the production of cholesterol in humans. There are two classes of this enzyme. Class I enzymes are found in humans, and Class II in bacterial pathogens. Statins, such as lovastation (LVA), are excellent inhibitors of Class I HMG-CoA reductase, but much less effective on the Class II enzymes. A HMG-CoA reductase structure in complex with LVA from Pseudomonas mevalonii (Class II) was solved to investigate the basis for this difference.
Tabernero L, Rodwell VW, and C Stauffacher. 2003 Crystal Structure of a Statin Bound to a Class II Hydroxymethylglutaryl-CoA Reductase. J Biol Chem. 278: 19933-19338
|Mike McGinley: Carbonic Anhydrase
Carbonic anhydrase is an abundant family of isozymes that function within all the different kingdoms of life. These enzymes drive the rapid interconversion of carbon dioxide and water into bicarbonate and hydrogen ions (CO2 + H2O ◊ HCO3- + H+). This reaction is essential for various biochemical processes spanning a wide array of organisms (e.g. acid-base balance in animal blood, coral calcification, dark-reactions in photosynthesis). The active site of carbonic anhydrase utilizes a zinc prosthetic group that is coordinated by the imidazole rings of three histidine residues. The association of the active site with a water molecule further polarizes the hydrogen-oxygen bonds of water by shifting the electron density towards the oxygen. This destabilizes the molecule, allowing it to donate a single proton to a coordinating histidine residue. The enzyme then brings the resulting hydroxide ion into close proximity with a bound carbon dioxide molecules. Hydroxide, a strong nucleophile, rapidly reacts with the carbon dioxide to form bicarbonate. This model highlights the zinc prosthetic groups and the three histidine residues associated with the active site of the carbonic anhydrase II isozyme.
Lindskog, S. 1997. Structure and mechanism of carbonic anhydrase. Pharmacology and Therapeutics. 74: 1-20.
|Dominique Cowart: Myoglobin
Myoglobin is structurally related to hemoglobin, and is used for oxygen storage and transport. It is typically found in mammalian skeletal or cardiac muscle tissues where continuous oxygen flow is required for metabolism. Myoglobin tends to be released from damaged muscle tissues, and can be a marker for detecting heart attacks in people with chest pain.
Myoglobin forms pigments responsible for the red color in meat and the redness partially comes from the charge of the iron ion and its attached oxygen. Sperm whale myoglobin is one of the most intensely studied proteins, both structurally and functionally, and myoglobin is also present in some species of invertebrates.
Kentaro Iwanami et al. 2006.
“cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins”. Comparative Biochemistry and Physiology, Part B 145, 249–256
Ascaffenburg: L-ascorbate peroxidase
Ascorbate peroxidase (APX) is an ezyme that is involved in the reduction of O2 to H2O by using ascorbate as a substrate. This enzyme is a hemoprotein (highlighted in video), with the haem cofactor being the site of oxidation-reduction. APX is characterized as a class 1 peroxidase similar to cytochromec peroxidase. APX in chloroplasts can be further broken down into thylakoid-bound APX and stroma-localized APX. It plays a vital role in the Mehler reaction which can generate ATP using light energy. The Mehler reaction involves the transport of electrons from the donor side of PSII to the reducing side of PSI. Organisms seem to use this reaction in order to keep intracellular O2 levels low in order to protect ezymatic nitrogenase. The Mehler reaction can also be used to examine reactive oxygen species evolution in algae. In the course of the Mehler reaction, hydrogen peroxide is created and is subsequently quenched by ascorbate peroxidase. Under stress, there may not be enough ascorbate peroxidase to keep with the production of H2O2, and thus an excess of H2O2 forms.
Asada, K. 2006. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiology. 141: 391-396
Ferredoxin-NADP+ Reductase (FNR) is responsible for the photoreduction of NADP+ within chloroplasts. FNR accepts electrons from Photosystem I (Ferredoxin) to reduce NADP+ to NADPH during non-cyclic electron transport. This step is the final in photosystem electron transport. FNR can carry two electrons at once, while Ferredoxin, the molecule which passes the electrons to FNR, can carry only one electron. Therefore FNR must interact with two Ferredoxin molecules to gain two electrons for the reaction. Two specific types of FNR have been discovered. One type of FNR participates in the ferredoxin-NADP reducing system by loosely binding to the surface of the thylakoid membrane, while the other type is anchored in the membrane. This VMD molecular model highlights the NADP+ molecules (green) and the FAD molecule (grey) bound to the FNR.
CJ Batie and H Kamin. 1984. Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex . J. Biol. Chem., Oct 1984; 259: 11976 - 11985.
Alcohol Dehydrogenase (ADH) catalyzes the transformation of glyceraldehyde to gycerol for sugar metabolism, and also allows for the consumption of alcohol. In bacteria and yeast, ADH plays a major role in fermentation. The pyruvate that is formed via glycolysis is converted to acetaldehyde and carbon dioxide, and the acetaldehyde is then reduced to ethanol by an ADH. This step is necessary to regenerate NAD+. ADH contains two zinc ions (Zn 2+). One of which is crucial for the operation of the enzyme: it is located at the catalytic site and holds the hydroxyl group of the alcohol in place.
Only in certain bacteria and some yeast have iron been found in their ADH (not shown in video). Compared to the other enzymes of its family, this ADH is sensitive to oxygen.
Plapp, B.V., H. Eklund, T.A. Jones, C.I. Branden. 1983. Three-dimensional structure of isonicotinimidylated liver alcohol dehydrogenase. J.Biol.Chem. v258 pp. 5537-47, 1983