13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein.
13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.
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 / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes; D002417:Cattle; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D000623:Porphobilinogen Synthase / Q000378:metabolism*; D011485:Protein Binding
D002247:Carbon Isotopes; D000622:Aminolevulinic Acid; D000623:Porphobilinogen Synthase