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Acetyl-coenzyme A synthesis from methyltetrahydrofolate, CO, and coenzyme A by enzymes purified from Clostridium thermoaceticum: attainment of in vivo rates and identification of rate-limiting steps.

Department of Chemistry, University of Wisconsin, Milwaukee 53201.

ABSTRACT

Many anaerobic bacteria fix CO2 via the acetyl-coenzyme A (CoA) (Wood) pathway. Carbon monoxide dehydrogenase (CODH), a corrinoid/iron-sulfur protein (C/Fe-SP), methyltransferase (MeTr), and an electron transfer protein such as ferredoxin II play pivotal roles in the conversion of methyltetrahydrofolate (CH3-H4folate), CO, and CoA to acetyl-CoA. In the study reported here, our goals were (i) to optimize the method for determining the activity of the synthesis of acetyl-CoA, (ii) to evaluate how closely the rate of synthesis of acetyl-CoA by purified enzymes approaches the rate at which whole cells synthesize acetate, and (iii) to determine which steps limit the rate of acetyl-CoA synthesis. In this study, CODH, MeTr, C/Fe-SP, and ferredoxin were purified from Clostridium thermoaceticum to apparent homogeneity. We optimized conditions for studying the synthesis of acetyl-CoA and found that when the reaction is dependent upon MeTr, the rate is 5.3 mumol min-1 mg-1 of MeTr. This rate is approximately 10-fold higher than that reported previously and is as fast as that predicted on the basis of the rate of in vivo acetate synthesis. When the reaction is dependent upon CODH, the rate of acetyl-CoA synthesis is approximately 0.82 mumol min-1 mg-1, approximately 10-fold higher than that observed previously; however, it is still lower than the rate of in vivo acetate synthesis. It appears that at least two steps in the overall synthesis of acetyl-CoA from CH3-H4folate, CO, and CoA can be partially rate limiting. At optimal conditions of low pH (approximately 5.8) and low ionic strength, the rate-limiting step involves methylation of CODH by the methylated C/Fe-SP. At higher pH values and/or higher ionic strength, transfer of the methyl group of CH3-H4folate to the C/Fe-SP becomes rate limiting.

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