SYNTHESIS, CHARACTERIZATION, AND ENZYMATIC CONVERSION OF NONHYDROLYZABLE ANALOGS OF PROPIONYLCOENZYME-A

We describe the synthesis of three novel analogues of propionyl-coenzy me A, in which the sulfur atom has been replaced by methylene, ethylen e, and thiomethylene, respectively. All three analogues, propionyl-det hia(carba)-CoA (1), propionyl-dethia(dicarba)-CoA (2), and S-(2-oxobut anyl)-CoA (3) were characterized by H-1 and P-31 NMR spectroscopy and FAB mass spectrometry. Propionyl-CoA-oxaloacetate transcarboxylase fro m Propionibacterium shermanii accepted the novel analogues as substrat es and carboxylated them to the corresponding methylmalonyl-CoA analog ues (4-6). The latter were further converted into the succinyl-CoA ana logues by the coenzyme-B-12-dependent methylmalonyl-CoA mutase from th e same organism. The succinyl-CoA analogues, succinyl-dethia(carba)-Co A (7), succinyl-dethia(dicarba)-CoA (8), and 4-carboxy(2-oxobutanyl)-C oA (9) were obtained on a preparative scale and their Michaelis consta nts (K-m) with methylmalonyl-CoA mutase were determined to be 0.136, 2 .20, and 0.132 mM, respectively (K-m for succinyl-CoA is 0.025 mM). Th e V-max values for 7, 8, and 9 are 1.1, 0.013, and 0.0047 mu mol min(- 1) U-1, respectively (V-max for succinyl CoA is 1.0). The utility of t he novel coenzyme A analogues in enzyme mechanistic studies is discuss ed.