About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Abstract

Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate a-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on NE of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-'H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, k:, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J BioZ Chem 260: 1 1 1 15-1 1 122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (kO.03) M" s". Using the Eigen equation, this figure yields a pK, of 9.1 f 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, JBiochem 67:291-295). The mechanistic significance of these results is discussed.

The catalytic role of tyrosine 254 in flavocytochrome b2 (l‐lactate dehydrogenase from baker's yeast)

“…Therefore, Tyr129 (Fl b 2 Tyr254) would only bind to the hydroxyl oxygen in the transition state. Additional evidencein favour of a carbanion mechanism has also been published for Fl b 2 [18–20] and another member of the family [21].…”

l‐Lactate dehydrogenation in flavocytochrome b2

“…Among the arguments in support of the PA mechanism is the reported p K a value of 9 in reduced Fcb2 for the group that abstracts the substrate Hα [14]. This value has been considered too low to be that of the reduced FMN N5 atom, which has been estimated to be ∼ 20 for free flavin [15].…”

X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with d-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation Mechanism

“…However, mechanistic alternatives have been discussed extensively over the last three decades. [2][3][4][5][6][7][8] These chemical mechanisms are illustrated in Fig. 1.…”