JBC, Vol. 250, Issue 1, 261-270, Jan, 1975
Electron paramagnetic resonance and water proton relaxation rate studies of formyltetrahydrofolate synthetase-manganous ion complexes. Evidence for involvement of substrates in the promotion of a catalytically competent active site
D. H. Buttlaire, G. H. Reed and R. Himes
Conformational properties of the active site of formyltetrahydrofolate
synthetase from Clostridium cylindrosorum have been examined by EPR
spectroscopy and by solvent proton relaxation rate (PPR) studies of
manganous complexes with the enzyme. Ternary enzyme-Mn-nucleotide complexes
give EPR spectra which are very similar to those for the binary
Mn-nucleotide complexes. However, upon addition of tetrahydrofolate to form
the quaternary complexes, enzyme-MnADP-tetrahydrofolate and enzyme
MnATP-tetrahydrofolate the EPR line shapes are changed substantially.
Spectra for the quaternary complexes exhibit narrow line widths, and the
splitting patterns are characteristic of a slightly asymmetric electronic
environment for the bound Mn(II). Addition of formate to the ADP quatenary
complex induces a further significant narrowing of the EPR line widths,
although in the absence of tetrahydrofolate, formate does not influence the
EPR spectrum for the enzyme-MnADP species. Both Pi and nitrate cause
changes in the EPR patterns for the higher complexes of the enzyme which
involve both ADP and tetrahydololate. However, the Pi effect is not
influenced by the presence of formate whereas the characteristic effect of
nitrate is potentiated only when formate is present. EPR sectra for the
thernary complex with the beta, gamma-methylene analog of ATP App(CH2)p
differ significantly from spectra for the binary App(CH)p complex is not
influenced by further additions of tetrahydrofolate and of tetrahydorfolate
and formate. The failure of spectra for the App(CH)p complex to respond to
additions of the other substrates for the reaction is in marked contrast to
the behavior found for the natural nucleotide substrates and is tentatively
attributed to the lack of a protein-mediated interaction between the
nucleotide and tetrahydrofolate binding sites in the analog complex. The
frequency dependence of solvent PRR in the presence of the various
complexes allows an estimate of the correlation times for electron-nuclear
dipolar interaction and thereby the extent of hydration of the bound Mn(II)
among the various complexes..
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