Reactivity of the thiol groups of Escherichia coli phosphofructo-1-kinase.
Modification of Escherichia coli phosphofructo-1-kinase (6-phosphofructokinase; EC 2.7.1.11) with several thiol modifying reagents led to a pseudo-first-order loss of activity that was associated with the modification of a single cysteine residue, identified as the cysteine at position 119 in the protein sequence. This cysteine was protected from reaction with vinyl pyridine, bromopyruvate, and dithionitrobenzoic acid by the substrate, fructose-6-P. In the crystal structure of the highly homologous phosphofructokinase from Bacillus stearothermophilus, cysteine 119 is sufficiently distant from the catalytic site to exclude a direct steric inhibition of the binding of substrate as a mechanism of inactivation for the modification. Thus, the inhibition is unlikely to be a direct one but to be the result of interference with the conformational change that is associated with fructose-6-P binding. A second thiol, position 283, was shown to be protected from reaction when the enzyme was in its native conformation. In contrast to the previously published sequence for the E. coli enzyme six cysteines as opposed to seven have been found both in enzyme from strain LE392 and in enzyme produced by a plasmid that was derived from pLC 16-4. The position in question, 75, was identified as phenylalanine.
Banas
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Department of Biological Chemistry and Structure, University of Health Sciences, Chicago Medical School, IL 60064.
Gontero
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B
Drews
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VL
Johnson
S L
SL
Marcus
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Kemp
R G
RG
D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology; D005636:Fructosephosphates / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D005636:Fructosephosphates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D010732:Phosphofructokinase-1; D003545:Cysteine