/sup 13/C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase
/sup 13/C NMR has been used to observe the equilibrium complex of (5,5-/sup 2/H,5-/sup 13/C)-5-aminolevulinate ((5,5-/sup 2/H,5-/sup 13/C)ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. (5,5-/sup 2/H,5-/sup 13/C)ALA (chemical shift 46.9 ppm in D/sub 2/O) was prepared from (5-/sup 13/C)ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction (5,5-/sup 2/H,5-/sup 13/C)ALA forms (2,11,11-/sup 2/H,2,11-/sup 13/C)PBG (chemical shifts 116.2 ppm for C/sub 2/ and 34.2 ppm for C/sub 11/ in D/sub 2/O). For the complex formed between (5,5-/sup 2/H,5-/sup 13/C)ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies and imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C/sub 2/ is -2.8 ppm from the free signal and C/sub 11/ is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively. To aid in interpretation of these shifts, the /sup 13/C NMR chemical shifts of PBG were investigated as functions of pH and a variety of organic solvents. The observed shifts of bound PBG are not consistent with simple protonation/deprotonation of PBG nor with changes that can be duplicated by solvation by simple organic solvents.
- Research Organization:
- Univ. of Pennsylvania School of Dental Medicine, Philadelphia (USA)
- OSTI ID:
- 7163821
- Journal Information:
- Biochemistry; (United States), Vol. 27:12
- Country of Publication:
- United States
- Language:
- English
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/sup 13/C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein
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Related Subjects
AMINOLEVULINIC ACID
NUCLEAR MAGNETIC RESONANCE
STEREOCHEMISTRY
HYDRO-LYASES
CARBON 13
CHEMICAL SHIFT
DEUTERIUM COMPOUNDS
SCHIFF BASES
SUBSTRATES
ZINC COMPOUNDS
AMINO ACIDS
CARBON ISOTOPES
CARBON-OXYGEN LYASES
CARBOXYLIC ACIDS
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
IMINES
ISOTOPES
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
RESONANCE
STABLE ISOTOPES
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