This study contributes toward understanding the mechanism of catalytic formation of mixed ketones in an attempt to improve their selectivity vs symmetrical ketones. A pulsed microreactor placed inside a gas chromatograph–mass spectrometer instrument was used to identify the source of carbonyl group and quantify its distribution among products of zirconia-catalyzed cross-ketonization reaction of a mixture of carboxylic acids, with the carbonyl group of one of the acids selectively labeled by 13C. A concept of enolic and carbonyl components in the ketonization mechanism was introduced to distinguish the sources of alkyl and acyl groups, respectively. The least branched acid was found to be the predominant source of CO2, the essential byproduct of ketonization. Thus the least branched acid is the preferred source of the alkyl group of the cross-ketone product, while the most branched acid provides the acyl group. Increased branching at the α carbon next to the carbonyl group decreased the reactivity of both the enolic and the carbonyl components. Following a pseudo first order kinetic analysis, the relative reaction rates for a common enolic component with a pair of different carbonyl components were measured by the method of competing reactions to obtain mechanistic insights. The distinction between two possible paths in the cross-ketonization mechanism was characterized quantitatively by assessing the difference in activation energies; the results obtained were explained by the steric effect of substituents. On the basis of detailed kinetic analysis, the rate-limiting step most likely occurs after the enolic component activation.

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Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.

Supporting information includes Table S1 on gas phase analysis and related details of the analytical protocol used.

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that "actin binds enzymes", which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.

This perspective addresses efficiency and selectivity of high-temperature lignin liquefaction processes conducted in various reaction media as sub-and supercritical fluids. The challenges in efficient and selective production of high-value organic monomers from lignin are reviewed critically, along with analytical protocols essential for their accurate recovery after lignin degradation. The current approaches targeting the formation of phenolic monomers from lignin are discussed in terms of their repolymerization, a process that decreases the reaction selectivity and yield of the dominant phenolic monomers. The potential to solve this grand challenge is analyzed in terms of acid and/or protic cosolvent application, reduction of the reaction temperature, "quenching" of the reactive lignin depolymerization intermediates, and presence of heterogeneous catalysts, such as zeolites, metals, and metal oxides, sulfides, and phosphides.

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give two CO 2 molecules. The gene for cyanase is part of the cyn operon, which includes cynT and cynS, encoding carbonic anhydrase and cyanase, respectively. Carbonic anhydrase functions to prevent depletion of cellular bicarbonate during cyanate decomposition (the product CO 2 can diffuse out of the cell faster than noncatalyzed hydration back to bicarbonate). Addition of cyanate to the culture medium of a ⌬cynT mutant strain of E. coli (having a nonfunctional carbonic anhydrase) results in depletion of cellular bicarbonate, which leads to inhibition of growth and an inability to catalyze cyanate degradation. These effects can be overcome by aeration with a higher partial CO 2 pressure (M. B. Guilloton, A. F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P. M. Anderson, and J. A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). The question considered here is why depletion of bicarbonate/CO 2 due to the action of cyanase on cyanate in a ⌬cynT strain has such an inhibitory effect. Growth of wild-type E. coli in minimal medium under conditions of limited CO 2 was severely inhibited, and this inhibition could be overcome by adding certain Krebs cycle intermediates, indicating that one consequence of limiting CO 2 is inhibition of carboxylation reactions. However, supplementation of the growth medium with metabolites whose syntheses are known to depend on a carboxylation reaction was not effective in overcoming inhibition related to the bicarbonate deficiency induced in the ⌬cynT strain by addition of cyanate. Similar results were obtained with a ⌬cyn strain (since cyanase is absent, this strain does not develop a bicarbonate deficiency when cyanate is added); however, as with the ⌬cynT strain, a higher partial CO 2 pressure in the aerating gas or expression of carbonic anhydrase activity (which contributes to a higher intracellular concentration of bicarbonate/CO 2 ) significantly reduced inhibition of growth. There appears to be competition between cyanate and bicarbonate/CO 2 at some unknown but very important site such that cyanate binding inhibits growth. These results suggest that bicarbonate/CO 2 plays a significant role in the growth of E. coli other than simply as a substrate for carboxylation reactions and that strains with mutations in the cyn operon provide a unique model system for studying aspects of the metabolism of bicarbonate/CO 2 and its regulation in bacteria.Cyanase (EC 4.3.99.1) catalyzes the reaction of cyanate with bicarbonate to give two molecules CO 2 (15):The synthesis of cyanase in Escherichia coli is induced by addition of cyanate to the growth medium (2). The gene for cyanase is part of the cyn operon, which includes three genes in the order cynT, cynS, and cynX, encoding carbonic anhydrase, cyanase, and a hydrophobic protein of unknown function, respectively (11,(30)(31)(32)(33). The function of carbonic anhydrase appears to be to prevent depletion of cellular bicarb...

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