Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III
Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer to total PBG consumption resulted in low yields of uroporphyrinogen I(uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane but was different from that of a 2-(hydroxymethyl)bilane. The /sup 13/C NMR spectra of incubations carried out with (11-/sup 13/C)PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. A protonated uro'gen I structure for this intermediate was ruled out by incubations using (2,11-/sup 13/C)PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.
- Research Organization:
- Universidad de Buenos Aires (Argentina)
- OSTI ID:
- 6864067
- Journal Information:
- Biochemistry; (United States), Vol. 27:13
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
LYASES
CONFIGURATION INTERACTION
PORPHYRINS
BIOSYNTHESIS
NUCLEAR MAGNETIC RESONANCE
PYRROLES
ABSORPTION SPECTRA
CARBON 13
CARBON 14 COMPOUNDS
CHEMICAL SHIFT
DEAMINATION
HEME
LABELLED COMPOUNDS
PROTONS
RHODOPSEUDOMONAS
AZOLES
BACTERIA
BARYONS
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ELEMENTARY PARTICLES
ENZYMES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
RESONANCE
SPECTRA
STABLE ISOTOPES
SYNTHESIS
550601* - Medicine- Unsealed Radionuclides in Diagnostics
550201 - Biochemistry- Tracer Techniques