Methanol enzyme catalysis

An extremely interesting application of KIEs and theory has been elucidating the effect of enzyme catalysis on the structure of the SN2 transition state. Schowen and coworkers29 30 measured the secondary a-deuterium and a-carbon 12C/13C KIEs for the enzyme-catalyzed SN2 methyl transfer reaction between G-adenosylmethionine (Fig. 18) and 3,4-dihydroxyacetophenone in the presence of the rat-liver enzyme catechol O-methyltransferase (COMT) at 37°C (Fig. 19) and for the closely related, uncatalyzed Sn2 reaction between methoxide ion and G-methyldibenzothiophenium ion in methanol at 25°C (Fig. 20). The near maximum a-carbon KIEs of 1.09 + 0.02 for the enzyme-catalyzed SN2 reaction and 1.08 + 0.01 for the uncatalyzed Sn2 reaction were taken as evidence that both transition states were symmetric. However,... 

In the first reaction, intramolecular attack by the hydroxyl on the carboxyl carbonyl proceeds 5 X 108 times faster than the corresponding inter-molecular process (2). In the second reaction, the rate increases 107-fold upon switching the solvent from methanol to dimethylformamide (3). Obviously, the huge rate increases in these organic systems do not necessarily prove that similar effects are at work in enzymes. But to be suspicious is quite natural, and many people, too numerous to mention, have pointed out the possible relationship between enzyme catalysis and intramolecularity or solvation effects. 

Because zeolites can also be manufactured with various proportions of aluminate, a catalyst can be tailored to meet the exact requirement of the process. It is calculated that the medium-pore zeolite ZSM-5 (a), operating at 454 °C and lOOtorr (1.3 X 10" Pa) pressure of hexane, can crack more than 37 molecules per active site per minute. At 538 °C the turnover rises to over 300 molecules per minute per active site. Other catalytic processes - toluene disproportionation, xylene isomerization, and methanol conversion (see later) - operate even faster, with hexane isomerization showing a turnover of as much as 4 x 10 per minute per active site. This indicates that rates of catalytic reactions with zeolites equal or exceed rates for enzyme catalysis. 

The differences in the rate constant for the water reaction and the catalyzed reactions reside in the mole fraction of substrate present as near attack conformers (NACs).171 These results and knowledge of the importance of transition-state stabilization in other cases support a proposal that enzymes utilize both NAC and transition-state stabilization in the mix required for the most efficient catalysis. Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82%, 57%, and 1% of chorismate conformers were found to be NAC structures (NACs) in water, methanol, and the gas phase, respectively.172 The fact that the reaction occurred faster in water than in methanol was attributed to greater stabilization of the TS in water by specific interactions with first-shell solvent molecules. The Claisen rearrangements of chorismate in water and at the active site of E. coli chorismate mutase have been compared.173 It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared with the water reaction. 

Extending these ideas to enzymatic catalysis, Jiang et al. reported the use of protamine-silica hybrid microcapsules in combination with a host gel-like bead structure to encapsulate several enzymes individually in the enzymatic conversion of C02 to methanol [20]. They used a layer-by-layer (LbL) method where alternately charged layers were deposited on an enzyme-containing CaC03 core. The layers, however, were not polyelectrolytes, but protamine and silica (Scheme 5.6). 

Catalysis, mechanism of electrodic reactions, 1258 in redox reactions. 1275 and enzymes, 1287 Cathodic deposition, 1307 Cathode, 1050. 1348, 1359, 1361 Chandrasekaran, methanol oxidation, 1269 Chapman, 877... 

Kinetic studies tell how fast enzymes act but by themselves say nothing about how enzymes catalyze reactions. They do not give the chemical mechanism of catalysis, the step-by-step process by which a reaction takes place. Most of the individual steps involve the simultaneous breaking of a chemical bond and formation of a new bond. Consider a simple displacement reaction, that of a hydroxyl ion reacting with methyl iodide to give the products methanol and iodide ions. 

Poorly soluble substrates are likely to be inaccessible for the reaction with the enzyme. A similar scenario occurs in polymerization reactions, where the conversion results in low yields due to the lower solubility of the polymeric products. In this way, nonaqueous enzymology has emerged in the last years to further widen the versatility of the enzymatic catalysis. Solubility of hydrophobic compounds in organic solvents is usually orders of magnitude higher than in water. An example is anthracene, which is nearly insoluble in water (0.07 mg/L), whereas its solubility in solvents such as methanol and trichloromethane increases to a large extent (1 x 104 and 33 x 104 times higher, respectively). 

The different catalytic responses of peroxidase in dioxane and methanol versus acetone are intriguing. It is clear that the effects of water-miscible solvents on enzymatic catalysis are not equivalent and for the first time quantitative kinetic data have been obtained which highlight this. However, the cause of this effect remains unresolved. We are continuing and expanding this kinetic study to include other solvents, both water-miscible and immiscible, and other phenols. This future study will enable rational and quantitative approaches for peroxidase-catalyzed phenolic polymerizations to be based on optimal solvent and phenol choices. From a more fundamental standpoint, this work has shown that enzymes may be more active in organic media than in water as long as optimal conditions are employed. There is no reason to believe peroxidase is unique in this respect. 

The observed decreases in catalysis of the substituted enzymes may be a consequence of increased energy barriers due to the losses of transition state solvation. The effect seems to be mainly on "galactosylation" (k2). This is supported by the results of the nucleophilic competition studies which showed that the addition of methanol to the assay did not result in an increase in the kcat-Furthermore, the kcat values for each enzyme were quite different depending upon which substrate was used. This indicates that "galactosylation" (ka) was rate determining, and shows that this step was affected much more than "degalactosylation" (ks) by the changes in solvation of the planar transition state. 

Ammonia monooxygenase catalyzes also the oxidation of methane (Hyman and Wood, 1983) and carbon monoxide (Tsang and Suzuki, 1982) to methanol and carbon dioxide, respectively, in addition to the catalysis of the oxidation of ammonia. The oxidation of methane by the enzyme will be described again below in relation to the regulation of the methane formation by the ammonia-oxidizing bacteria. 

Fig. 3 Cyclic voltammetric analysis of the kinetics of an homogeneous redox enzyme reaction using a reversible one-electron mediator as the cosubstrate, taking as example the catalysis of the electrochemical oxidation of j8-D-glucose by glucose oxidase (6.5 pM) with ferrocene methanol as the cosubstrate at pH = 7 (ionic strength 0.1 M, temperature ...

Without exception, trace amounts of water are found necessary for catalysis it performs a structural role, in which hydrogen bonding maintains the active conformation of the enzyme molecule [28, 29, 31]. Addition of polar cosolvents (methanol, ethanol, w-butanol, iso-buiano, t rt-butanol and acetone)... 

Biodiesel production from com oil can also be carried out in enzymatic catalysis. In a study using immobilized lipase enzyme (Novozym 435) as a catalyst, it was reported that 81.3% fattj acid methyl ester content was obtained at 15% enzyme load, 60°C temperatures and 10 MPa pressure in 4 horns (Ciftci Temelli 2013). Com oil was also transesterified with methanol by injecting it into a supercritical carbon dioxide stream in the presence of immobilized lipase enzyme. Fatty acid methyl ester yield was observed as greater than 98% (Meher et al., 2006). 

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