PubMed · 9700607
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E Bonioli, A Di Stefano, V Peri, U Caruso, R Cerone, E Lamantea, F Taroni, C Bellini. 1998. Fumarate hydratase deficiency.. https://doi.org/10.1023/a%3A1005379330187
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Overall activation volumes for multistep reactions are not usually pressure independent. The present investigation gives a quantitative description of this effect under Theory. Simple relations are obtained which can easily be applied to experimental data and which allow more insight into the dynamics of enzyme reactions. This is demonstrated under Experimental Application for the conversion of fumarate to L-malate catalyzed by the enzyme fumarase. The volume profile of this reaction indicates a pulsation of the enzyme molecule during catalysis. The appendix discusses the question whether Eyring's transition-state theory is an appropriate basis for investigations of this kind.
Stereochemical and product analyses have been studied in our continuing work on the bioprocessing of fluorinated substrate analogues. The hydration pathways of the fumarase-catalyzed reaction on fluorofumarate lead to a product distribution of L-threo-beta-fluoromalate to oxalacetate of 1 to 16. The beta-fluoromalate product has not been previously reported. Oxalacetate formation from the initial product, alpha-fluoromalate, an alpha-fluorohydrin, proceeds by way of a direct nonenzymic decomposition path (as opposed to collapse to the enol of oxalacetate with subsequent tautomerization). Difluorofumarate is hydrated to an alpha-fluorohydrin, alpha, beta-difluoromalate, which decomposes to 3(S)-fluorooxalacetate trapped by in situ malate dehydrogenase mediated reduction to L-threo-beta-fluoromalate (2R,3S). L-threo-Fluoro[2-3H]-malate is a slow substrate for the reverse reaction as measured by labilization of 3H while the erythro isomer is barely detectable. The pathways responsible for this volatilization are discussed. Acetylenedicarboxylate hydration stereochemistry was also determined where the initial product of the reaction, the enol of oxalacetate, tautomerized and was trapped by enzymic reduction to L-malate.
Fumarase (L-malate hydro-lysase E.C.4.2.1.2) was purified from the thermophilic bacteria Bacillus stearothermophilus NU-10 (optimum growth temperature 62-63 degrees C) and Thermus X-1 (optimum growth temperature 70 degrees C). The furmarase from Thermus X-1 is slightly more thermostable and has an "optimum" catalytic reaction temperature of 83 degrees C as compared to 81 degrees C for the B. stearothermophilus enzyme. Increased thermostability of these fumarases permitted an examination of the properties of the enzyme catalyzed reaction at temperatures higher than had previously been possible with the furmarases from mesophilic bacteria or higher plant and animal sources. Beyong the observed thermostability of the thermophilic fumarases, the catalytic properties of thermophilic fumarases were very similar to those observed with bacterial fumarase or the well characterized pig heart fumarase (effect of temperature on substrate affinities, pH optimum, substrate inhibition by fumarate, and Haldane relationship). These similarities suggest that thermophilic enzymes may be useful in the general study of enzyme reaction mechanisms.