An investigation of the substrate specificity of jack bean urease (urea amidohydrolase, EC 126.96.36.199) has revealed that both nitrourea and semicarbazide are substrates for the enzyme. Nitrourea at pH 5.0 has a kcat of 12.4 sec-1 and a Km of 135 mM. The corresponding parameters for semicarbazide at pH 5 and pH 7 are 50.3 sec-1, 29.6 sec-1 and 60 mM, 60 mM (cf. urea, 597 sec-1, 3.28 mM). N-methylurea, methyl carbamate, ethyl carbamate, carbamoyl azide, arginine, acetohydroxamic acid and carbamylcholine are demonstrated not to be substrates within varying degrees of tolerance.
Contrary to the report of Jespersen (1975), the final products of the urease-catalysed hydrolysis of urea in both Tris and citrate buffers are carbonic acid and ammonia. Jespersen's measurements may be explained by non-enzymatic reactions of both Tris and citrate with one or other of the products.
An extensive investigation of the physical properties of jack bean urease leads to the value 622,000 daltons for its molecular weight. While this value differs markedly from the accepted molecular weight of 483,000 daltons, all measurements in this laboratory are entirely consistent with the higher value. The value for the molecular weight of urease was greatly influenced by nonideality effects, and incomplete allowance for this, together with minimal studies of the partial specific volume and of the diffusion coefficient, led to the earlier, erring, values. The value for v̄ is 0.74 cm3g-1 and for the diffusion coefficient, 3.08 x 10-7 cm2sec-1. The work reported here also points up the paucity of reliable comparative data for proteins of high molecular weight.
A large variety of procedures has been used to investigate the active site of jack bean urease. All these studies reinforce the centrality of nickel ion in the mechanism of action of the enzyme. Experiments showed that neither a covalent urease-acetohydroxamic acid complex nor a covalent acyl-enzyme existed. There exists also the clear possibility that an active component of the mechanism of action of this enzyme is a nickel-bound hydroxide ion or water molecule. A generally useful procedure for investigating the presteady state of enzyme-catalysed reactions is partially developed and leads to the conclusion that the first product of the urease-catalysed hydrolysis of urea is the iminol of carbamic acid, rather than carbamic acid itself.