JBC, Vol. 252, Issue 7, 2262-2270, Apr, 1977
Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes
C. B. Ireland and P. G. Schmidt
Nuclear magnetic relaxation methods were used to investigate the
interaction of the inhibitor succinate with aspartate transcarbamylase from
Escherichia coli. Over the pH range 7 to 9, the dissociation constant for
succinate remains less than the inhibitor concentration used for most of
this work (0.05 M). As a result, the enzyme predominantly exists in a
single "gross" conformational state. Succinate binding to this enzyme state
(generally known as the R form) parallels the behavior seen previously with
the isolated catalytic subunit (Beard, C. B., and Schmidt, P.G. (1973)
Biochemistry 12, 2255-2264). The pH and temperature dependence of succinate
proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl
phosphate, is interpreted in terms of a binding mechanism involving three
forms of the enzyme, differing by their states of protonation. The least
protonated form of the enzyme does not interact with succinate, the singly
protonated species binds succinate to form a rapidly dissociating complex,
and the doubly protonated species undergoes a conformational isomerization
upon succinate binding, yielding a slow exchange complex. Relaxation data
provide sufficient information to determine pKa values of 7.2 and 8.9 for
two ionizing groups, as well as the dissociation constant for succinate in
the fast exchange complex, Kd =1.6 X 10(-2) M. Rate constants for the
forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1,
respectively, indicate a significantly slower reverse rate from that
obtained in the earlier NMR study of the isolated catalytic subunit. In
experiments where the succinate concentration was varied, the relaxation
rates showed sigmoidal binding of that ligand to the fast exchange complex
above pH 9.1, (a) indicating cooperative binding of succinate, and (b)
suggesting that above pH 9.1, the system cannot be characterized by a
single dissociation constant, ionization constant, or relaxation effect.
CTP and ATP were tested for their ability to affect succinate binding to
the fast exchange complex. Heterotropic interactions were observed for CTP
but not for ATP. Addition of low concentrations of the transition state
analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl
phosphate-succinate complex sharply decreased the relaxation rate,
indicating that the measurements are sensitive only to succinate bound
specifically to the active site.
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