The role of the C-terminal tail of flavocytochrome b2.
A flavocytochrome b2 (L-lactate dehydrogenase) mutant was constructed in which the C-terminal tail (23 amino acid residues) had been deleted (Gly-489----Stop). This tail appears to form many intersubunit contacts in the tetrameric wild-type protein, and it was expected that its removal might lead to the formation of monomeric flavocytochrome b2. The isolated tail-deleted mutant enzyme (TD-b2), however, was found to be tetrameric (Mr 220,000). TD-b2 shows Km and kcat. values (at 25 degrees C and pH 7.5) of 0.96 +/- 0.06 mM and 165 +/- 6 s-1 respectively compared with 0.49 +/- 0.04 mM and 200 +/- 10 s-1 for the wild-type enzyme. The kinetic isotope effect with [2-2H]lactate as substrate seen for TD-b2, with ferricyanide as electron acceptor, was essentially the same as that observed for the wild-type enzyme. TD-b2 exhibited loss of activity during turnover in a biphasic process. The rate of the faster of the two phases was dependent on L-lactate concentration and at saturating concentrations showed a first-order deactivation rate constant, kf(deact.), of 0.029 s-1 (at 25 degrees C and pH 7.5). The slower phase, however, was independent of L-lactate concentration and gave a first-order deactivation rate constant, ks(deact.), of 0.01 s-1 (at 25 degrees C and pH 7.5). This slower phase was found to correlate with dissociation of FMN, which is one of the prosthetic groups of the enzyme. Thus fully deactivated TD-b2, which was also tetrameric, was found to be completely devoid of FMN. Much of the original activity of TD-b2 could be recovered by re-incorporation of FMN. Thus the C-terminal tail of flavocytochrome b2 appears to be required for the structural integrity of the enzyme around the flavin active site even though the two are well separated in space.
White
S A
SA
Department of Chemistry, University of Edinburgh, U.K.
Black
M T
MT
Reid
G A
GA
Chapman
S K
SK
D004252:DNA Mutational Analysis; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000502:physiology*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007773:Lactates / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
D005415:Flavins; D007773:Lactates; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)