Abstract
The biosynthesis of D-glucosamine-6-phosphate by l-Glutamine: d-fructose-6-phosphate aminotransferase in the liver of the rat is irreversibly inactivated by injection of N-acetyl-6-diazo-5-oxo- l-norleucine, Duazomycin A. The course of inactivation parallels the disappearance of UDP-N-acetyl-hexosamine from the liver. Injection of glucosamine with Duazomycin A completely protects the enzyme against inactivation, without protecting enzymes involved in the biosynthesis of purine and cytosine nucleotides that are sensitive to Duazomycin A. The injection of glucosamine either 2 or 4 hr after Duazomycin A reduces the lag in appearance of aminotransferase activity without altering the ultimate rate of what appears to be resynthesis, since the process is inhibited by puromycin and dl-ethionine; the time required to regain 50% of the activity is 13 to 20 hr. Crude and partially purified extracts containing the aminotransferase are irreversibly inactivated by either Duazomycin A or 6-diazo-5-oxo- l-norleucine; the rate of inactivation by the latter is dependent upon its concentration over the range 1 × 10 −6 to 3 × 10 −4 m (K m = 8 × 10 −5 m ) . Both l-glutamine and UDP-N-acetylglucosamine reduce the rate of inactivation of the enzyme by 6-diazo-5-oxo- l-norleucine in vitro; a combination of both compounds is more effective than either alone, and evidence is presented that the two protective compounds act at different sites on the enzyme. The protective effects have been demonstrated in a crude extract and with a partially purified enzyme. Glucose-6-phosphate, fructose-6-phosphate and other amino sugars and nucleotide compounds related to UDP-N-acetyl-glucosamine do not protect against inactivation by 6-diazo-5-oxo- l-norleucine. The protective effect of UDP-N-acetylglucosamine in vitro appears to explain the protective effect of glucosamine in vivo. The implications of these findings are discussed with respect to the nature of the interaction between the enzyme, its two substrates, its feedback inhibitor (UDP-N-acetylglucosamine) and the inactivator, 6-diazo-5-oxo- l-norleucine. This system provides a means of studying the synthesis of a specific constitutive enzyme under controlled conditions in the liver.
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