Biosynthesis of porphyrins and corrins. 2. Isolation, purification, and NMR investigations of the porphobilinogen-deaminase covalent complex
The procedures for the generation of enzyme-substrate complexes from labeled porphobilinogens ((2,11-/sup 13/C)PBG and (2,6,11-/sup 3/H)PBG) with deaminase and the methods employed for their purification are described. Use of /sup 13/C NMR failed to detect the substrate bound to the enzyme, suggesting that the line width must be inordinately large. The complex was found to disproportionate with time when stored at 25 degrees C. However, enzyme-bound uroporphyrinogen I (uro'gen I) was detected, both in the intact protein and in the oligopeptides from tryptic digestion and peptide mapping. The first detection of an enzyme-substrate complex by /sup 3/H NMR is described for (/sup 3/H)PBG and deaminase. The line widths of the observed resonances were found to be extremely large and dependent upon temperature, giving chemical shifts that suggest the involvement of a sulfhydryl group as the nucleophilic enzyme group that binds the substrate. The catalytic competence of this complex was also demonstrated by displacing bound (/sup 3/H)PBG with unlabeled PBG. During the resultant formation of (3H)uro'gen I, a transient low-intensity signal was detected that has been tentatively assigned to the highly reactive azafulvene species, proposed in several mechanistic schemes for porphyrin biosynthesis.
- Research Organization:
- Texas AandM Univ., College Station
- OSTI ID:
- 5570049
- Journal Information:
- Biochemistry; (United States), Vol. 4
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
AMIDINASES
PURIFICATION
PORPHYRINS
BIOSYNTHESIS
BIOCHEMICAL REACTION KINETICS
CARBON 13
COVALENCE
NUCLEAR MAGNETIC RESONANCE
SUBSTRATES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
CARBON ISOTOPES
CARBOXYLIC ACIDS
ENZYMES
EVEN-ODD NUCLEI
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAGNETIC RESONANCE
NON-PEPTIDE C-N HYDROLASES
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
SYNTHESIS
400101* - Activation
Nuclear Reaction
Radiometric & Radiochemical Procedures
550201 - Biochemistry- Tracer Techniques