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/sup 13/C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

Journal Article · · Biochemistry; (United States)
OSTI ID:6075557
;

/sup 13/C NMR has been used to observe the equilibrium complex of (4-/sup 13/C)-5-aminolevulinate ((4-/sup 13/C)ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. (4-/sup 13/C)ALA (chemical shift = 205.9 ppm) forms (3,5-/sup 13/C)PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of (4-/sup 13/C)ALA and (/sup 15/N)ALA was used to assign the 121.0 and 123.0 ppm resonances to C/sub 5/ and C/sub 3/, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and (4-/sup 13/C)ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approx. 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 < 10 s/sup -1/, which is consistent with the turnover rate of the enzyme. For the complex formed from (4-/sup 13/C)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 approx. 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with /sup 113/Cd 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 /sup 113/Cd-/sup 13/C coupling was observed.

Research Organization:
Univ. of Pennsylvania School of Dental Medicine, Philadelphia
OSTI ID:
6075557
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:14; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
AMINOLEVULINIC ACID
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CADMIUM 113
CADMIUM COMPOUNDS
CADMIUM ISOTOPES
CADMIUM OXIDES
CARBON 13
CARBON ISOTOPES
CARBON-OXYGEN LYASES
CARBOXYLIC ACIDS
CHALCOGENIDES
CHEMICAL BONDS
CHEMICAL SHIFT
ENZYME ACTIVITY
ENZYMES
EVEN-ODD NUCLEI
IMINES
INTERMEDIATE MASS NUCLEI
ISOMERIC TRANSITION ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
NITROGEN 15
NITROGEN ISOTOPES
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDES
OXYGEN COMPOUNDS
PURIFICATION
RADIOISOTOPES
REACTION INTERMEDIATES
RESONANCE
SCHIFF BASES
SPECTRA
STABLE ISOTOPES
YEARS LIVING RADIOISOTOPES