The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase.
Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the Kms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.
Hirasawa
M
M
Department of Chemistry and Biochemistry, Texas Tech. University, Lubbock, TX 79409-1061, USA.
Hurley
J K
JK
Salamon
Z
Z
Tollin
G
G
Markley
J L
JL
Cheng
H
H
Xia
B
B
Knaff
D B
DB
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry*; D017033:Anabaena / Q000737:chemistry; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002384:Catalysis; D004579:Electron Transport; D005288:Ferredoxins / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism; D017433:Protein Structure, Secondary; D018724:Spinacia oleracea / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D000596:Amino Acids; D005288:Ferredoxins; D007656:Ketoglutaric Acids; D005973:Glutamine; D014364:Tryptophan; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin); D001974:Bromosuccinimide; D010269:Paraquat