Mutation to glutamine of histidine 373, the catalytic base of flavocytochrome b2 (L-lactate dehydrogenase).
Flavocytochrome b2 catalyzes the two-electron oxidation of L-lactate. Reducing equivalents are transferred first to FMN then to heme b2 in the same subunit, finally to cytochrome c or a non-physiological acceptor. The enzyme's three-dimensional structure, when analyzed in the light of existing mechanistic knowledge, suggested that His 373 is the active site base which initiates the substrate chemical transformation by abstracting the lactate alpha-proton. We report here the properties of a mutant enzyme with glutamine substituted histidine at position 373. The mutated enzyme preparations show a 10(4)-fold decrease in catalytic activity. We find that most of this residual activity can be eliminated by treatments with: 1) fluoropyruvate, an affinity label for His 373; and 2) 2- hydroxy-3-butynoate, a suicide reagent which normally forms an adduct with FMN but in this case leaves the bulk of the prosthetic group intact. Furthermore, although spectral titrations do not detect any binding of oxalate, this reagent inhibits the mutant enzyme with the same kinetic behaviour as for the wild-type enzyme. We conclude that the enzyme preparations contain about 1 in 10(4) molecules of wild-type flavocytochrome b2; this is probably due to codon misreading during biosynthesis. Thus the H373Q enzyme displays at most 10(5)-fold less activity than the wild-type enzyme. We report values for the spectrally determined binding constants of sulfite, pyruvate and D-lactate for the mutant enzyme. Finally, we show that 2,6-dichlorophenol indophenol, which is a 10-fold more sensitive routine electron acceptor than ferricyanide, accepts electrons only from heme b2 and not from the flavin.
Gaume
B
B
CNRS-URA 1461, Hôpital Necker, Paris, France.
Sharp
R E
RE
Manson
F D
FD
Chapman
S K
SK
Reid
G A
GA
Lederer
F
F
D015086:2,6-Dichloroindophenol; D001483:Base Sequence; D002384:Catalysis; D004579:Electron Transport; D005973:Glutamine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D042844:L-Lactate Dehydrogenase (Cytochrome); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D017186:Titrimetry
D005973:Glutamine; D006639:Histidine; D015086:2,6-Dichloroindophenol; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)