A-Acetylomithine

Several of the key enzymes in the acetylated pathway have been detected in plants. These include acetyl-CoArglutamate iV-acetyltransferase (Morris and Thompson, 1975, 1977 Staub and Denes, 1966), A-acetylglutamate 5-phosphotransferase (Farago and Denes, 1967 Cybis and Davis, 1974 McKay and Shargool, 1977), and A -acetylomithine 2-oxoglutarate aminotransferase (Dougall and Fulton, 1967). A model for the pathway of CIT synthesis in nodules of Alnus is presented in Fig. 11. 

Fig. 5. A Time course of the reduction in activity of acetylomithine S-transaminase by treatment of arginine-grown cells of the argR strain 961 of Escherichia cott with 0.4 M Mg + plus L-arginine hydrochloride (0.1 mg/ml), at 37° with aeration. The transaminase activity is determined [8] in extracts. B Restoration of acetylomithine 8-transaminase activity. An extract, prepared after treatment of cells wdth Mg + plus arginine for 2 hours, is kept at 0° and assayed at intervals. The activity of the extract is plotted as a percentage of that of an untreated-control extract (which remains approximately constant). From Leisinger et al. [67].

Three relevant enz3une activities have been demonstrated in E. coli extracts. These are an acetylase which forms JV-acetylglutamate from glutamic acid and acetyl CoA 129), a transaminase which forms N -acetylornithine 116), and acetylomithinase which hydrolyzes the N -acetylomithine to ornithine 116). It is highly significant that the last named enz3une was absent in extracts of a mutant blocked between iV -acetylomithine and ornithine 116). 

This is cited as evidence that molds and vertebrates synthesize ornithine from glutamic y-semialdehyde (131,132), while E. colt and related bacteria utilize the acetylornithine pathway. This interpretation has been contested by Davis (f). He points out that Neurospora may be impermeable to N -acetylomithine or that a block might occur after this compound in the auxotrophs studied. 

This enzyme primes the cycle with acetylglutamate. Once primed, the acetylor-nithine formed functions as the acetyl donor to complete the cycle. The transfer of the acetyl group from acetylomithine is carried out by the second enzyme, A -acetyl-L-omithineiL-glutamate iV-acetyltransferase (EC 2.3.1.35). 

Ornithine acetyltransferase has been partially purified from Chlamydomonas (Staub and Denes, 1966) and Chlorella (Morris and Thompson, 1975). The enzyme from Chlamydomonas was purified 60-fold and exhibited a broad pH optimum from 7.5 to 9. The values (pH 7.5) for glutamate and acetylomithine were 13 and 5.5 ta.M, respectively. The enzyme was specific for L-gluta-mate but would use other acetyl donors. The enzyme was not assayed for acetyl-CoA acetyltransferase activity. The reaction was reversible with a =... 

The enzyme from Chlorella possesses both acetyl-CoA acetyltransferase and acetylomithine acetyltransferase activity although the acetyl-CoA-dependent activity was more labile (Morris and Thompson, 1975). The enzyme purified over 180-fold had a pH optimum between 8 and 8.5. The values for glutamate, acetyl-CoA, and acetylomithine were 3, 3.2, and 0.2 mM, respectively. The enzyme was inhibited by arginine (A = 0.94 mM). Again, activity was unstable and no cofactor was required. The acetyl-CoA-dependent activity was stabilized by thiols and reduced by para-chloromercuribenzoate (PCMB) while the acetylomithine-dependent activity was not affected. These results suggest that free thiol is essential for activity of the acetyl-CoA acetyltransferase. 

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