Detection of carbamate as a product of the carbamate kinase-catalyzed reaction by stopped flow spectrophotometry.

It has been commonly assumed that carbamate kinnse (EC 2.7.2.2, ATP : carbamate phospllotransferase) catalyzes the transfer of a phosphate group between 1TP and the carbamate ion. Evidence which has been put forward supporting this belief is the observation that freshly dissolved ammonium carbamate is a much better substrate for the enzyme than freshly dissolved ammonium carbonate at pH 9.4 (1) or pH 8.5 (a), an effect abolished by the addition of carbonic anhydrase (1) or by the aging of the substrate solutions (2). These observations could be explained equally well on the assumption that carbon dioxide, and not bicarbonate, is the true substrate for the enzyme since it is known (3) that carbamate ion decomposes directly to carbon dioxide, not bicarbonate. If carbamate is the true substrate for the enzyme, by the priuciple of microscopic reversibility, it ought to be the immediate product of the reverse reaction between carbamyl phosphate and SDP. With the aid of stopped flow spectrophotometry we have developed a method for the det’ection of carbamate ion in aqueous solution. The method depends on the instability of carbamate at neutral pH. If a solution containing carbamate ion is brought rapidly to plT 7, it decomposes in a reaction associated with a rise in pI-1.