Purification and characterization of 2-enoyl-CoA reductase of Mycobacterium smegmatis.
2-Enoyl-CoA reductase was purified to homogeneity for the first time from the crude extract of Mycobacterium smegmatis. Its molecular weight was estimated to be 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted as an electron donor for the reduction of 2-enoyl-CoA, while NADPH did not. The Km value for NADH was 21.3 microM. On the other hand, NAD inhibited the reaction for competing against NADH, as the Ki value for NAD was 47 microM. Among the enoyl-CoAs used as substrates, those having C10-C16 were found to be most suitable substrates for the purified reductase in terms of both apparent Km and Vmax values. The enzyme was strongly inhibited, however, when the concentration of the C16-substrate was over 50 microM. The enzyme had almost no activity towards substrates having less than C8. When NAD3H was used as an electron donor to 2-dodecenoyl-CoA in the presence of the purified reductase, only laurate was tritiated as the product. Diacetyl and phenylglyoxal, agents that react specifically with arginine, inactivated the reductase in a time- and concentration-dependent manner during the preincubation. These results suggest that some arginine residues in the reductase protein are involved in the enzyme activity.
D003931:Diacetyl / Q000494:pharmacology; D044943:Fatty Acid Desaturases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008970:Molecular Weight; D009161:Mycobacterium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010658:Phenylglyoxal / Q000494:pharmacology
D009243:NAD; D044943:Fatty Acid Desaturases; D003931:Diacetyl; D010658:Phenylglyoxal