The role of the C-terminal tail of flavocytochrome b2

• Published in: Biochemical journal 1989-11, Vol.263 (3), p.849-853
• Main Authors: White, S A, Black, M T, Reid, G A, Chapman, S K
• Format: Article
• Language: English
• Subjects: DNA Mutational Analysis; Flavin Mononucleotide - metabolism; Flavins - metabolism; Kinetics; L-Lactate Dehydrogenase (Cytochrome); L-Lactate Dehydrogenase - physiology; Lactates - metabolism; Models, Molecular; Molecular Weight; Oxidation-Reduction; Protein Conformation; Saccharomyces cerevisiae - enzymology; Structure-Activity Relationship
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2630849

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.