3 Carbamate Kinase

Abstract

Publisher Summary This chapter discusses the synthesis of carbamyl phosphate (CAP) when ATP, ammonium carbamate or carbonate, and magnesium are the only substrates utilized; this synthesis is called “CKase” activity. The synthesis of CAP (in the presence or absence of an acyl-glutamate) from glutamine or ammonia, HCO 3 - , and 2 moles of MgATP per mole of CAP formed is referred to as “CAP synthetase” activity. The major glutamine utilizing synthetase can also form CAP from carbamate or ammonium carbonate. One of the enzymes of Escherichia coli previously referred to as “CKase” may be a synthetase, and the presumed kinase of Neurospora , which provides CAP for arginine biosynthesis, appears now to be a CAP synthetase. Cell extracts might contain any combination of these activities (CKase, CAP synthetase, and disrupted CAP synthetase), the distinction among which is in their stoichiometry. The chapter discusses the molecular properties—purification, composition, size, subunit structure, and stability—and effect of some sulfhydryl reagents. Purified CKases are unstable when in solution in dilute buffers, even at low temperatures. P -Hydroxymercuribenzoate inhibits the CKase from Streptococcus faecalis ; at 10 μM concentration, almost 90% inhibition of the forward and backward reactions is observed. This effect is partially reversed by a 50 M excess of cysteine with respect to p -hydroxymercuribenzoate. The chapter also discusses the carbamate kinase catalytic reactions, the specificity and cofactors, assays—design of assays for the forward and reversible reactions—thermodynamics, kinetics and catalytic mechanisms, function and relation to other enzymes, and the metabolite control of activity. In resting cultures, citrulline rather than ornithine is the main product of the breakdown of arginine. This is probably a result of the equilibrium constant of the ornithine transcarbamylation reaction, which strongly favors citrulline synthesis. In such a system, the reaction would proceed in the reverse direction only if one or both of the products are removed from the medium.