New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.
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).
Jordan
P A
PA
Department of Chemistry, University of Wyoming, Laramie, WY 82071-3838, USA.
Bohle
D S
DS
Ramilo
C A
CA
Evans
J N
JN
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D002826:Chorismate Mutase / Q000737:chemistry* / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014443:Tyrosine
D001426:Bacterial Proteins; D008670:Metals; D003035:Cobalt; D014443:Tyrosine; D008345:Manganese; D010728:Phosphoenolpyruvate; D003300:Copper; D007501:Iron; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D002826:Chorismate Mutase