Linear Eyring Plots Conceal a Change in the Rate-Limiting Step in an Enzyme Reaction

Machado, Teresa F. G.; Gloster, T.M.; Silva, Rafael G. da

doi:10.1021/acs.biochem.8b01099

Cited by 19 publications

(51 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is in reasonable agreement with experimental values, and captures the higher energy barrier reflected in the lower k cat for the mesophilic enzyme ( k cat = 2.6 ± 0.2 s –1 for Pa HBDH and k cat = 0.21 ± 0.01 s –1 for Ab HBDH). 20 The average values of Δ H ‡ calculated within the rigid-rotor harmonic oscillator approximation are overestimated, likely owing to the lack of proper thermal sampling of enzyme and solvent configurations. Nonetheless, the calculated average difference in Δ H ‡ between the two HBDHs is identical, within statistical error, to the experimental difference in Δ H ‡ .…”

Section: Results and Discussionmentioning

confidence: 99%

“…In addition, the reported exponential averages, as recommended by Cooper et al, 70 are in reasonable agreement with experimental values, showing the computational model captures the distinct thermodynamics of activation found experimentally for Pa HBDH and Ab HBDH. 20 …”

Section: Results and Discussionmentioning

confidence: 99%

“…Successful mutation was confirmed by DNA sequencing (MRC PPU DNA Sequencing and Services). All four mutant proteins were produced exactly as reported for the respective wild-type HBDH (WT-HBDH) 20 and analyzed for exact mass by electrospray ionization-mass spectrometry (ESI-MS). Initial rates of acetoacetate reduction catalyzed by all HBDH mutants were measured at 10 °C under the same conditions as the wild-type enzymes in the presence of varying concentrations of one substrate and a fixed, saturating concentration of the other.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Thus, one must be vigilant as to which step in the catalytic cycle is represented by kcat when studying enzyme kinetics as a function of temperature. Changing the thermal energy in such a system can influence either the chemical events, conformational movements, or both [9].…”

Section: Introductionmentioning

confidence: 99%

High catalytic rate of the cold-activeVibrioalkaline phosphatase depends on a hydrogen bonding network involving a large interface loop

Hjörleifsson

Helland

Magnusdottir

et al. 2020

Preprint

View full text Add to dashboard Cite

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part of cold-adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large-loop, that hovers over the active site of the other monomer. It presumably has a role in VAP high catalytic efficiency that accompanies extremely low thermal stability. We designed several different mutagenic variants of VAP with the aim of removing inter-subunit interactions at the dimer interface. Breaking the inter-subunit contacts from one residue in particular (Arg336) caused diminished temperature stability of the catalytically potent conformation and a drop in catalytic rate by a half. The relative B-factors of the R336L crystal structure, compared to the wild-type, confirmed increased surface flexibility in a loop on the opposite monomer, but not in the large-loop. Contrary to expectations, the observed reduction in stability with an expected increase in dynamic mobility resulted in reduced catalytic rate. This contradicts common theories explaining high catalytic rates of enzyme from cold-adapted organisms as being due to reduced internal cohesion bringing increased dynamic flexibility to catalytic groups. The large-loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl-group affinity (inactive).

show abstract

“…Thus, one must be vigilant as to which step in the catalytic cycle is represented by k cat when studying enzyme kinetics as a function of temperature. Changing the thermal energy in such a system can influence either the chemical events, conformational movements, or both [9].…”

mentioning

confidence: 99%

The high catalytic rate of the cold‐activeVibrioalkaline phosphatase requires a hydrogen bonding network involving a large interface loop

et al. 2020

View full text Add to dashboard Cite

show abstract

Linear Eyring Plots Conceal a Change in the Rate-Limiting Step in an Enzyme Reaction

Cited by 19 publications

References 31 publications

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry

High catalytic rate of the cold-activeVibrioalkaline phosphatase depends on a hydrogen bonding network involving a large interface loop

The high catalytic rate of the cold‐activeVibrioalkaline phosphatase requires a hydrogen bonding network involving a large interface loop

Contact Info

Product

Resources

About