Characterization of the active site of chloroperoxidase using physical techniques
Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.
- Research Organization:
- Illinois Univ., Urbana (USA)
- OSTI ID:
- 5349047
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
BARYONS
DEUTERIUM
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIGANDS
LIGHT NUCLEI
MAGNETIC RESONANCE
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PEROXIDASES
PROTEINS
PROTONS
RECEPTORS
RESONANCE
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES