ARCHIVES

OF

lsocitrate

BIOCHEMISTRY

AND

174, 695-704 (1976)

Lyase from Pseudomonas indigofera: pH Dependence Catalysis and Binding of Substrates and Inhibitors’ JOHN

Program

BIOPHYSICS

in Biochemistr,y

E. ROGERS’

AND

BRUCE

and Bioph.ysics in the Depurtment Pullman, Washington Received

November

of

A. MCFADDEN:’

of Chemistry, 99163

Washington

State lJniuersit.v,

7, 1975

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomowas dependent on two dissociable groups (pK,,‘s of 6.9 and 8.6). The pH nns indigofern dependence of the pK, for succinate, a product of isocitrate cleavage, implied that a dissociable group (pK,, of 6.0) on the enzyme functions in binding succinate. The pK,‘s for maleate and itaconate (succinate analogs) were similarly pH dependent. The pK, for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pK,‘s of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pK,,, for isocitrate and the pK, for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme ipK,, of 5.8). The pH dependence of the pK, for homoisocitrate was similar. In addition the K, for succinate and K,,, for isocitrate were dependent upon Mg’+ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofcra, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pK