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Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

ABSTRACT: Human aromatase is the cytochrome P450 catalyzing the conversion of androgens into estrogens in a three steps reaction essential to maintain steroid hormones balance. Here we report the capture and spectroscopic characterization of its compound I (Cpd I), the main reactive species in cytochromes P450. The typical spectroscopic transitions indicating the formation of Cpd I are detected within 0.8 s when mixing aromatase with meta-chloroperoxybenzoic acid. The estrogen product is obtained from the same reaction mixture, demonstrating the involvement of Cpd I in aromatization reaction. Site-directed mutagenesis is applied to the acid-alcohol pair D309 and T310 and to R192, predicted to be part of the proton relay network. Mutants D309N and R192Q do not lead to Cpd I with an associated loss of activity, confirming that these residues are involved in proton delivery for Cpd I generation. Cpd I is captured for T310A mutant and shows 2.9- and 4.4-fold faster rates of formation and decay, respectively, compared to wild-type (WT). However, its activity is lower than the WT and a larger amount of H₂ O₂ is produced during catalysis, indicating that T310 has an essential role in proton gating for generation of Cpd 0 and Cpd I and for their stabilization. The data provide new evidences on the role of threonine belonging to the conserved "acid-alcohol" pair and known to be crucial for oxygen activation in cytochromes P450.

SUBMITTER: Zhang C

PROVIDER: S-EPMC9366932 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

Zhang Chao C Gilardi Gianfranco G Di Nardo Giovanna G

Protein science : a publication of the Protein Society 20220901 9

Human aromatase is the cytochrome P450 catalyzing the conversion of androgens into estrogens in a three steps reaction essential to maintain steroid hormones balance. Here we report the capture and spectroscopic characterization of its compound I (Cpd I), the main reactive species in cytochromes P450. The typical spectroscopic transitions indicating the formation of Cpd I are detected within 0.8 s when mixing aromatase with meta-chloroperoxybenzoic acid. The estrogen product is obtained from the ...[more]

PMID: 36040260

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Project description:Alcohol dehydrogenase catalyzes the reversible transfer of a hydride directly from an alcohol to the nicotinamide ring of NAD+ to form an aldehyde and NADH, and the proton from the alcohol probably is transferred through a hydrogen-bonded system to the imidazole of His-48. Studies of the pH dependencies, and solvent and substrate isotope effects on the wild-type and the enzyme with His-48 substituted with Gln-48 were used to demonstrate a role for the proton relay system. The H48Q substitution increases affinities for NAD+ and NADH by ∼2-fold, suggesting that the overall protein structure is maintained. In contrast, catalytic efficiencies (V/Km) on ethanol and acetaldehyde and affinity for 2,2,2-trifluoroethanol are decreased by about 10-fold. The pH dependencies for catalytic efficiencies on ethanol and acetaldehyde (log V/Km versus pH), show pK values of about 7.5 for wild-type enzyme, but ethanol oxidation by H48Q ADH is essentially linear over the pH range from 5.5 to 9.2 with a slope of 0.47. Steady-state kinetics and substrate isotope effects suggest that the kinetic mechanism of H48Q ADH has become partly random for oxidation of ethanol. Both wild-type and H48Q ADHs have pH-independent isotope effects for oxidation (V1/Kb) of 1-butanol/1-butanol-d9 of 4, suggesting that hydride transfer is a major rate-limiting step. The pH dependence for butanol oxidation by wild type ADH shows a wavy profile over the pH range from pH 6 to 10, with a ∼2.3-fold larger V1/Kb in D2O than in H2O, an "inverse" isotope effect. The substrate isotope effect of 4 is not altered by the solvent isotope effect, suggesting concerted proton/hydride transfer. The solvent isotope effect can be explained by a ground state with a water bound to the catalytic zinc in the enzyme-NAD+ complex, and a transition state that resembles a complex with NADH and aldehyde. In contrast, the H48Q enzyme has a diminished inverse solvent isotope effect of ∼1.3 and an essentially linear pH dependence with a slope of log V1/Kb against pH of 0.49 for oxidation of 1-butanol, which together are consistent with a transition state where hydroxide ion directly accepts a proton from the 2'-hydroxyl group of the nicotinamide ribose in the proton relay system in the enzyme-NAD+-alcohol complex. The results support a catalytic role for His-48 in the proton relay system.

Project description:X-Ray crystallography shows that the hydroxyl group of Thr-45 in the fermentative alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae is hydrogen-bonded to the hydroxyl group of the alcohol bound to the catalytic zinc and is part of a proton relay system linked to His-48. The contribution of Thr-45 to catalysis was studied with steady state kinetics of the enzyme with the T45G substitution. Affinities for coenzymes decrease by only 2-4-fold, but the turnover numbers (V/Et) and catalytic efficiencies (V/KmEt) decrease 480-fold and 2900-fold for the oxidation of ethanol and 450-fold and 8400-fold for acetaldehyde reduction, respectively, relative to wild-type enzyme. Binding of NADH appears to require protonation of a group with a pK value of ∼7.4 in wild-type ADH1, but the pK value for T45G ADH1 appears to be less than 5. For wild-type enzyme, the pH dependencies for ethanol oxidation (V1/Et and V1/KbEt) are maximal above pK values of 7.0-7.7 and are attributed to the ionization of the alcohol or water bound to the catalytic zinc facilitated by His-48 in the enzyme-NAD+ complexes. For T45G ADH1, these pK values are shifted to 6.3. The reduction of acetaldehyde (V2/Et and V2/KpEt) modestly increases as the pH increases for wild-type and T45G enzymes. The removal of the hydroxyethyl group of Thr-45 disrupts the connection of the oxygen of ligands bound to the catalytic zinc with the proton relay system and formation of productive catalytic states. The conformational change of the enzyme and the exchange of ligands on the catalytic zinc can also be affected. Assignments of groups responsible for the pK values are discussed in the context of studies on other forms of horse liver and yeast ADHs. The substitutions with Ala-45 and Cys-45 in yeast ADH1 and the homologous substitutions with Ala-48 in horse and human liver ADHs also significantly decrease catalytic efficiency. Threonine or serine residues at this position in alcohol dehydrogenases are highly conserved and contribute substantially to catalysis.

Dataset Information

Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

Publications

Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

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