Study Of The Binding Of Substrate By The Phe557Val Mutant Soybean Lipoxygenase-1

Lipoxygenases are a class of enzymes which consist of non-heme iron dioxygenases that are produced by fungi, plants, and mammals and catalyze the oxygenation of polyunsaturated fatty acid substrates to unsaturated fatty acid hydroperoxide products. The unsaturated fatty acid hydroperoxide products are stereo- and regiospecific. One such lipoxygenase, soybean lipoxygenase-1 (SBLO-1), catalyzes the conversion of linoleate to 13-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) and a small amount of 9-hydroperoxy-10(E),12(Z)-octadecadienoate (9-HPOD). Although the structure of SBLO-1 is known and it is the most widely studied lipoxygenase, how it binds to substrate is still poorly understood. Two competing binding hypotheses that have been used to understand and explain the binding are the head first binding model and the tail first binding model. The head first binding model predicts linoleate binds with its polar carboxylate group in the binding pocket and the methyl terminus at the surface of the binding pocket. The tail first binding model predicts that linoleate binds with its methyl terminus end in the binding pocket and the polar carboxylate group at the surface of the binding pocket. Both binding models have been used in the explanation of previous work. In previous work the replacement of phenylalanine with valine has been performed to produce the phe557val mutant SBLO-1. The mutant SBLO-1 was then used in the enzymatic oxygenation of linoleate. With this mutant, the amount of 9-HPOD that is formed increases. This result has been interpreted using the head-first binding model in which the smaller valine residue allows linoleate to bind with the polar carboxylate group of linoleate interacting with arginine-707. The work presented in this thesis confirms the regiochemical results of the previous work and further tests the head-first binding model. If head-first binding occurs, the 9-HPOD is expected to have primarily S configuration. Utilizing chiral-phase HPLC, it was found that the 9-HPOD produced by the phe557val mutant SBLO-1 is primarily S, consistent with head-first binding. The head-first binding model was also tested using linoleyl dimethylamine (LDMA), which has been shown to be a good substrate for SBLO-1 at pH 7.0, where LDMA is thought to be positively charged. This model predicts that less of the 9-peroxide should be produced with this substrate. Through the use of gas chromatography/mass spectrometry, it was found that the conversion of LDMA by the phe557val mutant SBLO-1 resulted in the formation of a 46:54 mixture of the 13-peroxide:9-peroxide. The higher amount of 9-peroxide is the opposite of what is expected for the currently proposed model suggesting that the proposed model may not be entirely correct. The results thus far have been consistent with reverse binding but not with the proposed interaction of the polar end of the substrate with arginine-707.

Probing The Active And Allosteric Binding Sites Of Soybean Lipoxygenase-1

Soybean lipoxygenase-1 is a model for lipoxygenase activity. While the mechanism of oxygenation is understood, the substrate binding mechanism has not yet been elucidated. Two putative binding mechanisms are the ¿head-first¿ and ¿tail-first¿ models, in which the carboxy-terminus or the methyl terminus of the fatty acid substrate is inserted into the active site while the remainder of the molecule protrudes from the surface, respectively. Previous work has demonstrated that derivatization of fatty acid substrates with D-tryptophan increases active site affinity. It has also been shown that while polyunsaturated fatty acids are the natural substrates of lipoxygenases, monounsaturated fatty acids can be oxygenated at a much slower rate. Starting with a monounsaturated fatty acid, oleic acid, as a platform, the molecule N-oleoyl-D-tryptophan (ODT) was synthesized with the anticipation of it being a potent competitive substrate-analogue inhibitor that could be used to discern the substrate binding mechanism. Inhibition kinetics demonstrated that this molecule functions as a partially competitive inhibitor, through an unknown mechanism. The implication behind partially competitive inhibition is that substrate and inhibitor molecules can bind simultaneously to the enzyme, which alludes to the presence of an allosteric binding domain. To investigate the possibility of an inhibitor binding site on the non-catalytic subunit, limited proteolysis was used to cleave the subunits apart which should have eliminated inhibition. Interestingly, it was observed that at high substrate concentrations the inhibitor was completely ineffective, but at low substrate concentrations the inhibitor maintained its standard efficacy. A satisfactory explanation for these results has not yet been determined.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Synthesis Of Linoleate Analogs With Longer Alkyl Termini To Test For Proposed "Tail-First" Binding In SBLO-1

Lipoxygenases are nonheme-iron proteins that catalyze the oxygenation of polyunsaturated fatty acids to give conjugated diene hydroperoxides. For example, soybean lipoxygenase-1 (SBLO-1) converts linoleate into 13-(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13(S)-HPOD). Although the crystal structure of SBLO-1 has been determined, it is still unclear how the substrate binds at the active site. This absence of knowledge makes it difficult to understand the role of the enzyme during catalysis of the reaction. We hypothesize that SBLO-1 binds linoleate ¿tail-first¿, so that the methyl terminus is within a hydrophobic pocket deep within the enzyme. It is believed that the hydrophobic residue phenylalanine-557 at this site has stabilizing interactions with the terminal methyl group on linoleate. To test this hypothesis, we have developed a synthetic pathway that will yield linoleate analogs with longer fatty acid chains by 1 and 2 more carbons at the alkyl terminus. These substrates will be analyzed through kinetic assays done in combination with wild type SBLO-1 and mutants in which we have replaced phenylalanine-557 with valine.

When that tune runs through your head: A PET investigation of auditory imagery for familiar melodies

The present study used positron emission tomography (PET) to examine the cerebral activity pattern associated with auditory imagery forfamiliar tunes. Subjects either imagined the continuation of nonverbaltunes cued by their first few notes, listened to a short sequence of notesas a control task, or listened and then reimagined that short sequence. Subtraction of the activation in the control task from that in the real-tune imagery task revealed primarily right-sided activation in frontal and superior temporal regions, plus supplementary motor area(SMA). Isolating retrieval of the real tunes by subtracting activation in the reimagine task from that in the real-tune imagery task revealedactivation primarily in right frontal areas and right superior temporal gyrus. Subtraction of activation in the control condition from that in the reimagine condition, intended to capture imagery of unfamiliarsequences, revealed activation in SMA, plus some left frontal regions. We conclude that areas of right auditory association cortex, together with right and left frontal cortices, are implicated in imagery for familiartunes, in accord with previous behavioral, lesion and PET data. Retrieval from musical semantic memory is mediated by structures in the right frontal lobe, in contrast to results from previous studies implicating left frontal areas for all semantic retrieval. The SMA seems to be involved specifically in image generation, implicating a motor code in this process.

When that tune runs through your head: a PET investigation of auditory imagery for familiar melodies

The present study used positron emission tomography (PET) to examine the cerebral activity pattern associated with auditory imagery for familiar tunes. Subjects either imagined the continuation of nonverbal tunes cued by their first few notes, listened to a short sequence of notes as a control task, or listened and then reimagined that short sequence. Subtraction of the activation in the control task from that in the real-tune imagery task revealed primarily right-sided activation in frontal and superior temporal regions, plus supplementary motor area (SMA). Isolating retrieval of the real tunes by subtracting activation in the reimagine task from that in the real-tune imagery task revealed activation primarily in right frontal areas and right superior temporal gyrus. Subtraction of activation in the control condition from that in the reimagine condition, intended to capture imagery of unfamiliar sequences, revealed activation in SMA, plus some left frontal regions. We conclude that areas of right auditory association cortex, together with right and left frontal cortices, are implicated in imagery for familiar tunes, in accord with previous behavioral, lesion and PET data. Retrieval from musical semantic memory is mediated by structures in the right frontal lobe, in contrast to results from previous studies implicating left frontal areas for all semantic retrieval. The SMA seems to be involved specifically in image generation, implicating a motor code in this process.

Investigation of the Potential Energy Surface for the First Step in the Alkaline Hydrolysis of Methyl Acetate

The potential energy surface for the first step of the alkaline hydrolysis of methyl acetate was explored by a variety of methods. The conformational search routine within SPARTAN was used to determine the lowest energy am1 and pm3 structures for the anionic tetrahedral intermediate. Ab initio single point and geometry optimization calculations were performed to determine the lowest energy conformer, and the linear synchronous transition (lst) method was used to provide an initial structure for transition state optimization. Transition states were obtained at the am1, pm3, 3-21G, and 3-21 + G levels of theory. These transition states were compared with the anionic tetrahedral intermediates to examine the assumption that the intermediate is a good model for the transition state. In addition, the Cramer/Truhlar sm3 solvation model was used at the semiempirical level to compare gas phase and aqueous alkaline hydrolysis of methyl acetate.

Integrated Geophysical Investigation of the St. James Fault Complex: A Case Study

We noninvasively detected the characteristics and location of a regional fault in an area of poor bedrock exposure complicated by karst weathering features in the subsurface. Because this regional fault is associated with sinkhole formation, its location is important for hazard avoidance. The bedrock lithologies on either side of the fault trace are similar; hence, we chose an approach that capitalized on the complementary strengths of very low frequency (VLF) electromagnetic, resistivity, and gravity methods. VLF proved most useful as a first-order reconnaissance tool, allowing us to define a narrow target area for further geophysical exploration. Fault-related epikarst was delineated using resistivity. Ultimately, a high-resolution gravity survey and subsequent inverse modeling using the results of the resistivity survey helped to further constrain the location and approximate orientation of the fault. The combined results indicated that the location of the fault trace needed to be adjusted 53 m south of the current published location and was consistent with a north-dipping thrust fault. Additionally, a gravity low south of the fault trace agreed with the location of conductive material from the resistivity and VLF surveys. We interpreted these anomalies to represent enhanced epikarst in the fault footwall. We clearly found that a staged approach involving a progression of methods beginning with a reconnaissance VLF survey, followed by high-resolution gravity and electrical resistivity surveys, can be used to characterize a fault and fault-related karst in an area of poor bedrock surface exposure.

Investigation of Rudimentary Meta-Cognition in Lion-Tailed Macaques and Tufted Capuchins

Meta-cognition, or "thinking about thinking," has been studied extensively in humans, but very little is known about the process in animals. Although great apes and rhesus macaques (Macaca mulatta) have demonstrated multiple apparently meta-cognitive abilities, other species have either been largely ignored or failed to convincingly display meta-cognitive traits. Recent work by Marsh, however, raised the possibility that some species may possess rudimentary or partial forms of meta-cognition. This thesis sought to further investigate this possibility by running multiple comparative experiments. The goal of the first study was to examine whether lion-tailed macaques, a species that may have a rudimentary form of meta-cognition, are able to use an uncertainty response adaptively, and if so, whether they could use the response flexibly when the stimuli for which the subjects should be uncertain changed. The macaques' acquisition of the initial discrimination task is ongoing, and as such there were not yet data to support a conclusion either way. In the second study, tufted capuchins were required to locate a food reward hidden beneath inverted cups that sat on a Plexiglas tray. In some conditions the capuchins were shown where the food was hidden, in others they could infer its location, and in yet others they were not given information about the location of the food. On all trials, however, capuchins could optionally seek additional information by looking up through the Plexiglas into the cups. In general, capuchins did this less often when they were shown the food reward, but not when they could infer the reward's location. These data suggest capuchins only meta-cognitively control their information seeking in some conditions, and thus, add support to the potential for a rudimentary form of meta-cognition. In convergence with other studies, these results may represent early models for rudimentary meta-cognition, although viable alternative explanations still remain.

Geophysical Investigation of Subsurface Three-Dimensional Architecture of Icy Debris Fans With Ground Penetrating Radar in the Wrangell Mountains, Alaska

Icy debris fans have are newly-described landforms (Kochel and Trop, 2008 and 2012) as landforms developed immediately after deglaciation on Earth and similar features have been observed on Mars. Subsurface characteristics of Icy debris fans have not been previously investigated. Ground penetrating radar (GPR) was used to non-invasively investigate the subsurface characteristics of icy debris fans near McCarthy, Alaska, USA. The three fans investigated in Alaska are the East, West, and Middle fans (Kochel and Trop, 2008 and 2012) which below the Nabesna ice cap and on top of the McCarthy Creek Glacier. Icy debris fans in general are a largely unexplored suite of paraglacial landforms and processes in alpine regions. Recent field studies focused on direct observations and depositional processes. Their results showed that the fan's composition is primarily influenced by the type and frequency of depositional processes that supply the fan. Photographic studies show that the East Fan receives far more ice and snow avalanches whereas the Middle and West Fans receive fewer mass wasting events but more clastic debris is deposited on the Middle and West fan from rock falls and icy debris flows. GPR profiles and Wide-angle reflection and refraction (WARR) surveys consisting of both, common mid-point (CMP), and common shot-point (CSP) surveys investigated the subsurface geometry of the fans and the McCarthy Creek Glacier. All GPR surveys were collected in July of 2013 with 100MHz bi-static antennas. Four axial profiles and three cross-fan profiles were done on the West and Middle fans as well as the McCarthy Creek Glacier in order to investigate the relationship between the three features. GPR profiles yielded reflectors that were continuous for 10+ m and hyperbolic reflections in the subsurface. The depth to these reflections in the subsurface requires knowledge of the velocity of the subsurface. To find the velocity of the subsurface eight WARR surveys collected on the fans and on the McCarthy Creek glacier to provide information on variability of subsurface velocities. The profiles of the Middle and West fan have more reflections in their profiles compared to profiles done on the McCarthy Creek Glacier. Based on the WARR surveys, we interpret the lower energy return in the glacier to be caused by two reasons. 1) The increased attenuation due to wet ice versus drier ice and on the fan with GPR velocities >0.15m/ns. 2) Lack of interfaces in the glacier compared to those in the fans which are inferred to be produced by the alternating layers of stratified ice and lithic-rich layers. The GPR profiles on the West and Middle Fans show the shallow subsurface being dominated by lenticular reflections interpreted to be consistent with the shape of surficial deposits. The West Fan is distinguished from the Middle Fan by the nature of its reflections patterns and thicknesses of reflection packages that clearly shows the Middle fan with a greater thickness. The changes in subsurface reflections between the Middle and West Fans as well as the McCarthy Creek Glacier are thought to reflect the type and frequency of depositional processes and surrounding bedrock and talus slopes.