JBC, Vol. 252, Issue 6, 1957-1964, Mar, 1977
Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons
D. H. Buttlaire and M. Chon
The interactions of substrates with succinyl-CoA synthetase were
investigated by measuring the enhancement of the longitudinal water proton
relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The
binding of Mn(II) to the enzyme was investigated by EPR. The effects of
phosphorylating the enzyme on its interactions with Mn(II) and substrates
were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase
(E) at approximately four sites with a KD value of 0.14 mM, and the PRR
enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8.
The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four
sites with a KD value of 0.74 mM, and only a small change in epsilonb to
18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values
of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about
126 times more weakly to the enzyme than does Mn-ADP. PRR titrations
indicated that the values of epsilont for the ternary E-Mn-ADP and
(E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not
at all to (E-P)-Mn.Formation of the ternary complexes of CoA with E-Mn or
(E-P)-Mn could be followed by small but significant increases in the PRR
enhancement. No ternary complex with succinate could be detected since the
addition of succinate had no effect on the PRR enhancement. However, a
large decrease in enhancement, at least 2-fold, was observed upon addition
of both succinate and CoA. An increase in the PRR enhancement was produced
by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of
the dissociation constants for CoA from the quaternary
E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary
E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The
epsilon values for the quaternary and quinary complexes are 6.4 and 3.1,
respectively. The successive occupation of substrate binding sites of
succinyl-CoA synthetase produces alterations in the molecular dynamics or
in the conformation of the active site (or both), which are accompanied by
progressive decreases in the values of epsilon. Thus, the physical
parameter used in these studies relects the previously observed catalytic
properties of the enzyme system inasmuch as the catalytic function of
succinyl-CoA synthetase is potentiated by substrate binding, and catalytic
avtivity in partial reactions is maximized as binding sites are
successively occupied.
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