posted on 2001-06-22, 00:00authored byPeter A. Jordan, D. Scott Bohle, Cecilia A. Ramilo, Jeremy N. S. Evans
Metal binding properties for a series of metal-substituted forms of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, DAHPS(Tyr), have been followed by UV−vis and EPR spectroscopy.
The results show that there are two metal species present at pH = 7.0 and these are coordinated in a
distorted metal binding site with a mixed nitrogen and oxygen donor atom coordination set. There is no
spectroscopic evidence for strong M−S interactions in this system at any pH. Metal saturation occurs at
a substoichiometric ratio of 0.8−0.85 metal/monomer, and the binding trends mirror previously published
enzyme activity profiles. There is a conformational change for CuDAHPS under basic conditions, and
equivalent protein handling for apoDAHPS leads to apparent loss of metal binding ability. Addition of
the substrate PEP does not alter the UV−vis spectra, but there are small changes in the EPR spectra of
CuDAHPS(Tyr). Further addition of the substrate analogue A5P has no effect on either spectra. Taken
together, these results serve to link previous studies on enzyme activity with the recently determined
X-ray crystal structure for DAHPS(Phe) and represent the first detailed spectroscopic characterization of
the metal binding properties of DAHPS(Tyr).