A Kinetic Study of the Mechanism of Crystalline Carbamate Kinase

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, s20,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, Km carbamate = 80 µm, Kmatp = 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, Km carbamyl-p = 100 µm, Kmadp = 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.