Dataset Information

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

ABSTRACT: His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.

SUBMITTER: Tsai CL

PROVIDER: S-EPMC2526232 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

Tsai Chi-Lin CL Gokulan Kuppan K Sobrado Pablo P Sacchettini James C JC Fitzpatrick Paul F PF

Biochemistry 20070612 26

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is ...[more]

PMID: 17563122

Similar Datasets

Project description:D(--)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(--)-mandelate to phenylglyoxylate. D(--)-2-(Bromoethanoyloxy)-2-phenylethanoic acid ['D(--)-bromoacetylmandelic acid'], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(--)-Mandelate dehydrogenase was inactivated by D(--)-bromoacetylmandelate in a psuedo-first-order process. D(--)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(--)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(--)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(--)-bromoacetylmandelate reacts. D(--)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(--)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

Project description:The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.

Project description:The L-lactate-flavocytochrome b2-ferricyanide electron-transfer system from the yeast Hansenula anomala was investigated by rapid-reaction techniques. The kinetics of reduction of oxidized flavocytochrome b2 by L-lactate and L-[2H]lactate were biphasic both for flavin and haem prosthetic groups and at all concentrations tested. The first-order rate constants of the rapid and slow phases depended upon substrate concentrations, a saturation behaviour being exhibited. Substitution of the C alpha-H atom by 2H was found to cause appreciable changes in the rate constants for the initial reduction of flavin and haem (phase I), which were respectively about 3-fold and 2-fold less than with L-lactate. In contrast, no significant isotope effect was noted on the apparent reduction rate constants of the slow phase, phase II. Under steady-state conditions an isotope effect of 2.0 was found on the overall electron transfer from L-lactate to ferricyanide. These transient reduction results were discussed in terms of a kinetic model implying intra- and inter-protomer electron exchanges between flavin and haem b2, all of which have been experimentally described. Computer simulations indicate that the reaction scheme provides a reasonable explanation of the fast-reduction phase, phase I (in absence of acceptor). The pseudo-first-order rate constant for oxidation of reduced haem b2 in flavocytochrome b2 increased with increasing ferricyanide concentration in a hyperbolic fashion. The limiting value at infinite ferricyanide concentration, which was attributed to the intramolecular electron-transfer rate from ferroflavocytochrome b2 to the iron of ferricyanide within a complex, was 920 +/- 50 s-1 at pH 7.0 and 5 degrees C. Stopped-flow and rapid-freezing measurements showed haem b2 and flavin to be 90 and 44% oxidized respectively under steady-state conditions in presence of ferricyanide. Simulation studies were carried out to check the participation of the proposed reduction sequence in the overall catalytic reaction together with the role of reduced haem b2 (Hr) and flavin semiquinone (Fsq) as electron donors to ferricyanide. When the rate of the intramolecular electron-transfer exchange between Fsq and ferricyanide was adjusted to 200 s-1, simulated data accounted for molar activities defined under various conditions of L-lactate, [2H]lactate and ferricyanide concentrations. Simulation studies were extended to data obtained using cytochrome c as acceptor and reaction catalysed by Saccharomyces cerevisiae flavocytochrome b2. The differences in reactivity observed for Hr and Fsq with ferricyanide and cytochrome c were discussed in terms of redox potentials, electrostatic interactions, distances and accessibility of the participating groups.

Dataset Information

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

Publications

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets