13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase.
13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]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 an 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, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Jaffe
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EK
Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia 19104-6002.
Markham
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GD
D000622:Aminolevulinic Acid; D002244:Carbon / Q000032:analysis*; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D011162:Porphobilinogen; D000623:Porphobilinogen Synthase / Q000378:metabolism*; D012545:Schiff Bases; D012997:Solvents
D012545:Schiff Bases; D012997:Solvents; D002244:Carbon; D011162:Porphobilinogen; D000622:Aminolevulinic Acid; D003903:Deuterium; D000623:Porphobilinogen Synthase