| 1. |
- Machado, Teresa F. G., et al.
(författare)
-
Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry
- 2020
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:24, s. 15019-15032
-
Tidskriftsartikel (refereegranskat)abstract
- The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD(+):acetoacetate and NAD+:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.
|
|
| 2. |
- Prah, Alja, et al.
(författare)
-
How Monoamine Oxidase A Decomposes Serotonin : An Empirical Valence Bond Simulation of the Reactive Step
- 2020
-
Ingår i: Journal of Physical Chemistry B. - : AMER CHEMICAL SOC. - 1520-6106 .- 1520-5207. ; 124:38, s. 8259-8265
-
Tidskriftsartikel (refereegranskat)abstract
- The enzyme-catalyzed degradation of the biogenic amine serotonin is an essential regulatory mechanism of its level in the human organism. In particular, monoamine oxidase A (MAO A) is an important flavoenzyme involved in the metabolism of monoamine neurotransmitters. Despite extensive research efforts, neither the catalytic nor the inhibition mechanisms of MAO enzymes are currently fully understood. In this article, we present the quantum mechanics/molecular mechanics simulation of the rate-limiting step for the serotonin decomposition, which consists of hydride transfer from the serotonin methylene group to the N5 atom of the flavin moiety. Free-energy profiles of the reaction were computed by the empirical valence bond method. Apart from the enzymatic environment, the reference reaction in the gas phase was also simulated, facilitating the estimation of the catalytic effect of the enzyme. The calculated barrier for the enzyme-catalyzed reaction of 14.82 +/- 0.81 kcal mol(-1) is in good agreement with the experimental value of 16.0 kcal mol(-1), which provides strong evidence for the validity of the proposed hydride-transfer mechanism. Together with additional experimental and computational work, the results presented herein contribute to a deeper understanding of the catalytic mechanism of MAO A and flavoenzymes in general, and in the long run, they should pave the way toward applications in neuropsychiatry.
|
|
| 3. |
- Socan, Jaka, et al.
(författare)
-
Computer simulations explain the anomalous temperature optimum in a cold-adapted enzyme
- 2020
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Among cold-adapted enzymes, there are also examples that show an enigmatic inactivation at higher temperatures before unfolding of the protein occurs. Here, we analyze these phenomena by extensive computer simulations of the catalytic reactions of psychrophilic and mesophilic alpha -amylases. The calculations yield temperature dependent reaction rates in good agreement with experiment, and also elicit the anomalous rate optimum for the cold-adapted enzyme, which occurs about 15 degrees C below the melting point. This result allows us to examine the structural basis of thermal inactivation, which turns out to be caused by breaking of a specific enzyme-substrate interaction. This type of behaviour is also likely to be relevant for other enzymes displaying such anomalous temperature optima. Enzymes from organisms inhabiting cold environments (psychrophiles) have adapted to catalyzing chemical reactions at near freezing temperatures. Here - using molecular dynamics simulations - the authors analyze cold adaptation of psychrophilic alpha -amylase and provide the structural basis for its low anomalous temperature optimum: the increased mobility of a surface loop involved in substrate interaction.
|
|
| 4. |
- Åqvist, Johan, et al.
(författare)
-
Hidden conformational states and strange temperature optima in enzyme catalysis
- 2020
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 59:40, s. 3844-3855
-
Tidskriftsartikel (refereegranskat)abstract
- The existence of temperature optima in enzyme catalysis that occur before protein melting sets in can be described by different types of kinetic models. Such optima cause distinctly curved Arrhenius plots and have, for example, been observed in several cold-adapted enzymes from psychrophilic species. The two main explanations proposed for this behavior either invoke conformational equilibria with inactive substrate-bound states or postulate differences in heat capacity between the reactant and transition states. Herein, we analyze the implications of the different types of kinetic models in terms of apparent activation enthalpies, entropies, and heat capacities, using the catalytic reaction of a cold-adapted alpha-amylase as a prototypic example. We show that the behavior of these thermodynamic activation parameters is fundamentally different between equilibrium and heat capacity models, and in the alpha-amylase case, computer simulations have shown the former model to be correct. A few other enzyme-catalyzed reactions are also discussed in this context.
|
|
