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A Kinetic Study of the Mechanism of Crystalline Carbamate Kinase

From the ¹ From the Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706

A procedure involving precipitation with ammonium sulfate and chromatography on diethylaminoethyl cellulose is described for isolating crystalline carbamate kinase from Streptococcus faecalis. The crystalline enzyme sedimented as a single component, s_20,w = 4.1 S at 8.8 mg ml^-1, and showed only traces of two other components on electrophoresis of a 1% solution in starch gel. The enzyme bound 1 mole of adenosine diphosphate per 33,000 g of protein with a dissociation constant of 6.6 µm; the minimum molecular weight from the amino acid composition was found to be 31,000.

Double reciprocal plots of initial velocity with respect to substrate concentration were linear in a range of substrate concentrations encompassing the values of the Michaelis constants. In the forward direction: V = 92 moles/31,000 g x sec, K_{m carbamate} = 80 µm, K_{m atp} = 8 µm, and the dissociation constant of the binary complex, enzyme-adenosine triphosphate, is 70 µm. In the reverse direction: V = 730 moles/31,000 g x sec, K_{m carbamyl-p} = 100 µm, K_{m adp} = 50 µm, and the dissociation constant of the enzyme-ADP complex is 5 µm. The product inhibition patterns fit a mechanism in which a nucleotide is the first substrate to add and the last to dissociate from the enzyme and in which a ternary complex is formed. The rate-limiting steps are the dissociations of the nucleotides from the enzyme.

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