Urea Amidolyase

The ATP-dependent cleavage of urea, catalyzed by urea amidolyase, occurs in two steps: (a) urea + ATP + HC03 Mg*, K+ , b allophanate + ADP + Pi; and (b) allophanate + 2 HC03-+ 2 NH4+.The enzyme, purified approximately 150-fold from extracts of Candida utilis, contains high levels of covalently bound biotin and is very sensitive to inhibition by avidin.Furthermore, under controlled conditions, [r4C]biotin is incorporated covalently into the enzyme when its formation is induced.The biotin-free apoenzyme of urea amidolyase has been isolated from Saccharomyces cerevisiae grown under biotin-deficient conditions.This apoenzyme catalyzes the hydrolysis of allophanate but does not catalyze the net cleavage of urea.The in vitro conversion of the apoenzyme to the holoenzyme requires ATP, Mg++ ions, d-biotin, and a low molecular weight holoenzyme synthetase from S. cerevisiae.Chemically prepared d-biotinyl 5'-adenylate will substitute for d-biotin and ATP in this process.In a previous communication (l), WC presented preliminary evidence that urea amidolyase from Candida utilis catalyzes the cleavage of urea according to the following sequence of reactions: Urea + ATP + HCOs-t Mg++, K+ ' allophanate + ADP + Pi (1) Allophanate + 2 HCO3 + 2 NHa+ (2) Net, reaction: Urea + ATP Mg++, K+ HCOa-> HCO, + 2 NH,+ + ADP + Pi (3)Avidin, which is known to selectively bind biotin, inhibits