Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 323-332).

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

Polyunsaturated fatty acids (PUFA) comprise a significant portion of mammalian brain tissue, and are involved in neural signalling and cellular homeostasis. One brain PUFA, arachidonic acid, represents an attractive target for manipulation, with evidence suggesting it plays a role in the pathology of several neurological diseases. In this study, we fed rats a 15-week diet of an n-3 PUFA adequate or deprived diet, and then injected 3H arachidonic acid into the right lateral ventricle and measured its rate of loss over time. The half-life was 44 and 46 days for the n-3 PUFA adequate and deprived dietary groups, respectively. We compared the rate of loss with a predicted rate of loss (~45 days). We concluded that plasma unesterified AA is quantitatively a major source of brain phospholipid AA. Furthermore, we demonstrated selective regulation of brain PUFA by showing AA, unlike DHA, is not conserved in n-3 PUFA deprivation.

The reaction of dimethyldioxirane with a series of cis/trans-1,2-dialkylalkenes was carried out and produced the corresponding epoxides in high yield. As expected, the relative reactivity at 23 ºC of the cis-alkenes was at least 8-fold greater than that of the trans-counterparts with the magnitude of the relative reactivity increasing with increased steric bulk. Enhanced selectivity for cis- versus trans-alkene epoxidation was observed at lower temperatures. The reaction of dimethyldioxirane with selected alkenes was carried out in various solvent conditions (dried acetone:acetonitrile (1:9), dried acetone:methanol (1:9), dried acetone:carbon tetrachloride (1:9) and acetone:water (Xwater = 0.00, 0.01, 0.02, 0.03, 0.04, 0.05)) and produced the corresponding epoxides in high yield. The reactivity of dioxirane with simple di- and trialkylalkenes was enhanced as the polarity and hydrogen bonding capability of the solvent system were increased. Little to no change in reactivity was observed in the non-polar solvent system. Epoxidation of trisubstituted alkenes by dioxirane showed a greater rate enhancement in polar protic solvents compared to that for the epoxidation of the disubstituted alkenes. The epoxidation of an allylic alcohol by dimethyldioxirane showed a large increase in the non-polar solvent system compared to that in acetone. The reaction of dimethyldioxirane with the allylic alcohol also exhibited less of a rate increase in polar protic systems than its alkyl counterpart. Activation parameters for the epoxidation of cis/trans-1,2-dialkylalkenes by dioxirane in dried acetone and the previously mentioned solvent systems were determined using the Arrhenius method. In general, the ∆G‡ and ∆H‡ terms were greater for the reaction of dimethyldioxirane with trans-alkenes as compared to those for the corresponding cis-isomers regardless of solvent or alkyl steric bulk. The calculated ∆S‡ terms appeared essentially independent of steric bulk or solvent composition and were roughly identical, within experimental error, for all of the five cis/trans pairs. The ∆∆G‡ values, a comparison of the trans- to the cis-isomer data, yielded values of 1.2 to 1.8 kcal/mol in dried acetone for the five pairs of alkenes and appeared to be dependent on relative steric interactions. The ∆∆G‡ values for the epoxidation of cis/trans-alkenes carried out in solvents other than acetone showed no change from the value obtained in acetone. The experimental activation parameter data in dried acetone were consistent with predictions from ab initio calculations based on a spiro transition state model.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

Polyunsaturated fatty acids (PUFA) comprise a significant portion of mammalian brain tissue, and are involved in neural signalling and cellular homeostasis. One brain PUFA, arachidonic acid, represents an attractive target for manipulation, with evidence suggesting it plays a role in the pathology of several neurological diseases. In this study, we fed rats a 15-week diet of an n-3 PUFA adequate or deprived diet, and then injected 3H arachidonic acid into the right lateral ventricle and measured its rate of loss over time. The half-life was 44 and 46 days for the n-3 PUFA adequate and deprived dietary groups, respectively. We compared the rate of loss with a predicted rate of loss (~45 days). We concluded that plasma unesterified AA is quantitatively a major source of brain phospholipid AA. Furthermore, we demonstrated selective regulation of brain PUFA by showing AA, unlike DHA, is not conserved in n-3 PUFA deprivation.

Background: Knee joint motion and quadriceps activity play a crucial role in all lower limb tasks, especially those which are highly dynamic and weight-bearing. Due to anatomical differences between men and women such as height, leg length, and hip width, alignment and mechanics of the lower limb are different between males and females. An anatomical variable which is associated with alignment in the lower limb is the quadriceps muscle angle (q-angle). The purpose of this study is to determine if there is a relationship between q-angle, activity of the quadriceps and hamstring muscles and the kinetics and kinematics of the knee during stairclimbing. An investigation on the reliability of q-angle measurements was also made prior to the primary study. Methods: To test the interclass reliability of q-angle measurements, three individuals measured the q-angle on 20 subjects. The primary researcher measured the same twenty individuals on three separate days to determine intra-rater reliability. The primary study involved 10 male and 10 female subjects completing 20 stair-climbing trials (10 ascent, 10 descent). Kinematic and kinetic data were collected on the lower limbs as well as electromyography (EMG) on two quadriceps muscles and one hamstring muscle. Knee joint peak and occurrence of peak moments, average EMG amplitude and peak and occurrence of peak EMG were analyzed by gender and high and low q-angle. A two way analysis of variance (ANOVA) was used to test the statistical significance of each measured variable (alpha = 0.05). Results & Discussion: The inter-rater reliability for q-angle was low (0.27-0.78) but the intrarater reliability showed q-angle measurements to be very reliable (0.80-0.95). For study 2, it was found that females had increased vastus lateralis and vastus medialis peak EMG and average EMG amplitudes for stair ascent and descent compared to males. Furthermore, for descent only, females demonstrated having delayed occurrence of peak EMG for vastus lateralis and biceps femoris, and exhibited an increased peak knee extension moment and a decreased peak knee adduction moment compared to males. For q-angle, there was a significant difference found for biceps femoris occurrence of peak EMG during descent, with the high q-angle group having delayed occurrence of peak. For ascent, the high q-angle group had significantly increased average vastus lateralis EMG and an earlier occurrence of knee abduction moment. Q-angles were found to be higher for women compared to men. Conclusion: This study confirms that gender differences do exist in knee moment and thigh EMG parameters with stair ascent and stair descent. With the high incidence of significant findings for the quadriceps muscle, further investigation is warranted to determine if a relationship does exist between q-angle and knee joint function. It would also be recommended that hip mechanics be included in future studies due to the difference seen in adduction moments at the knee.

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

Diimide generated from the hydrazine/hydrogen peroxide/catalyst system can be used to hydrogenate unsaturated polymers in latex form. As an economical and environmentally benign alternative to the commercial processes based on hydrogen/transition metal catalysts, this method is of special interest to industry. This thesis provides a detailed description of the diimide hydrogenation process. Reaction kinetics, catalysts and gel formation mechanism have been investigated. <br /> <br /> Four main reactions and a mass transfer process form three parallel processes in this system: diimide is generated at the interface of the latex particles; diimide diffuses into the organic phase to saturate carbon-carbon double bonds; diimide may be consumed at the interface by hydrogen peroxide, and may also be consumed by the disproportionation reaction in the organic phase. The two side reactions contribute to the low hydrogenation efficiency of hydrogen peroxide. Slowing down hydrogen peroxide addition and using stable interfacial catalysts may totally suppress the side reaction in the aqueous phase. The actual catalytic activity of metal ions in the latex depends on the hydrogen peroxide concentration and the addition procedure of reactants. Cupric ion provides better selectivity for hydrogenation than ferric ion and silver ion do. Boric acid as a promoter provides improved selectivity for hydrogenation and faster diimide generation rate. The side reaction in the rubber phase results in low efficiency and gel formation. The rate constants of the four reactions in this system are estimated. <br /> <br />It is shown that the hydrogenation of nitrile rubber latex with an average particle diameter of 72 nm is mainly a reaction-controlled process. Diimide diffusion presents limitation upon hydrogenation at high hydrogenation degree range. Antioxidants can not effectively inhibit gel formation during hydrogenation. Hydrogenation of a core-shell latex with NBR as the shell layer should be able to achieve a higher efficiency, a higher degree of hydrogenation and a lower level of crosslinking.