Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli.
To characterize the methionine biosynthetic enzyme cystathionine gamma-synthase from Escherichia coli, we have constructed high copy number plasmids containing the metB structural gene but lacking the closely linked metJ regulatory gene. When cloned into an appropriate strain, these plasmids can direct the overproduction of cystathionine gamma-synthase such that about 10% of the soluble protein is this enzyme. An efficient purification scheme has been developed that has allowed us to obtain gram quantities of enzyme. The active form is a tetramer with subunits of about 40,000 daltons and one pyridoxal phosphate cofactor per monomer. The kinetic constants for several enzyme-catalyzed reactions were determined at 25 degrees C. The Km value for the elimination reaction with O-succinyl-L-homoserine was calculated to be 0.33 mM with maximal velocity of 460 min-1. The Km for the elimination (deamination) reaction with vinylglycine was 5.6 mM with maximal velocity of 900 min-1. The Km values for the replacement reaction were calculated to be 1.0 mM for O-succinyl-L-homoserine and 0.05 mM for L-cysteine with maximal velocity of 700 min-1. The enzyme shows an absorption band at 422 nm (epsilon = 8463 M-1 cm-1) attributable to the Schiff base form of the pyridoxal phosphate cofactor. Steady-state spectra of reaction complexes show appearance of new longer wavelength absorbing materials during reaction with O-succinyl-L-homoserine, vinylglycine, or vinylglycine and L-cysteine. Reaction with O-succinyl-L-homoserine and L-cysteine produces only a red shift and slight reduction of the band at 422 nm.
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710.
E L
EL
Holbrook
R C
RC
Greene
J H
JH
Krueger
D019757:Carbon-Oxygen Lyases; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000378:metabolism; D013053:Spectrophotometry
D004269:DNA, Bacterial; D046911:Macromolecular Substances; C022696:O-succinylhomoserine (thiol)-lyase; D015252:Deoxyribonucleases, Type II Site-Specific; C032823:GGTNACC-specific type II deoxyribonucleases; D008190:Lyases; D019757:Carbon-Oxygen Lyases