Glutaminase of Escherichia coli

Experiments are reported on the steady state kinetic behavior of Escherichia coli glutaminase. The ratios of rates of hydrolysis and hydroxylaminolysis are the same for acid, amide, ester, and thioester substrates of the enzyme, whereas the corresponding ratios for similar derivatives of acetic acid in nonenzymatic reactions vary over several orders of magnitude. The kcat for all substrates is constant between pH 3.5 and pH 5.6, but above pH 5.3 the Km values increase very rapidly with pH so that, in effect, the enzyme becomes inactive at about pH 5.8. Velocities of hydrolysis of several substrates are 1.1- to 1.5-fold faster in H2O than in D2O. The thermodynamic activation parameters have been determined for the hydrolysis of certain substrates. These results and those reported in the accompanying papers lead to the following conclusions. The Km values are dissociation constants for the ES complex. All substrates bind to the same ionic form of the enzyme, which is stabilized by the cooperative action of 4 or 5 acidic groups per active site. The hydrolysis of all substrates proceeds through a common intermediate, the formation of which is the slowest step in the catalytic cycle. The characteristics of the rate-determining step do not clearly indicate whether covalent reactions or noncovalent (conformational) processes are involved. If the latter is the case, then it is not possible with the steady state methods used to decide between an acylation-deacylation pathway and a single displacement mechanism.