| 1. |
- Patton, Gregory C., et al.
(author)
-
Cofactor mobility determines reaction outcome in the IMPDH and GMPR (beta-alpha)(8) barrel enzymes
- 2011
-
In: Nature Chemical Biology. - 1552-4450 .- 1552-4469. ; 7, s. 950-958
-
Journal article (peer-reviewed)abstract
- Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP(star) as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 angstrom from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.
|
|
| 2. |
- Sevastik, Robin
(author)
-
Quantum chemical modeling of enzymatic reactions : applications to the tautomerase superfamily
- 2008
-
Licentiate thesis (other academic/artistic)abstract
- In this thesis, quantum chemical methods are used to investigate enzymatic reaction mechanisms. The Density functional theory, in particular the hybrid B3LYP functional, is used to model two enzymes belonging to the tautomerase superfamily; 4-Oxalocrotonate Tautomerase (4-OT) and cis-Chloroacrylic Acid Dehalogenase (cis-CAAD). The methodology is presented and new mechanistic insights for the two enzymes are discussed. For 4-OT, two different models are built and the potential energy curves are computed. This allows the methodology to be evaluated. The results give new insight into the energetics of the 4-OT reaction, indicating that the charge-separated intermediate is quite close in energy to the reactant species. The models also make it possible to perform in silico mutations to investigate the role of active site groups. Excellent agreement is found between the calculations and site-directed mutagenesis experiments, further substantiating the validity of the models. For cis-CAAD, the uncatalyzed reaction is first considered and excellent agreement is found between the calculated barrier and the measured rate constant. The enzymatic reaction is then studied with a quite large active site model and a reaction mechanism is proposed.
|
|
| 3. |
- Sevastik, Robin, et al.
(author)
-
Quantum chemical modeling of enzymatic reactions : the case of 4-oxalocrotonate tautomerase
- 2007
-
In: Bioorganic chemistry (Print). - : Elsevier BV. - 0045-2068. ; 35:6, s. 444-457
-
Journal article (peer-reviewed)abstract
- The reaction mechanism of 4-oxalocrotonate tautomerase (4-OT) is studied using the density functional theory method B3LYP. This enzyme catalyzes the isomerisation of unconjugated alpha-keto acids to their conjugated isomers. Two different quantum chemical models of the active site are devised and the potential energy curves for the reaction are computed. The calculations support the proposed reaction mechanism in which Pro-1 acts as a base to shuttle a proton from the C3 to the C5 position of the substrate. The first step (proton transfer from C3 to proline) is shown to be the rate-limiting step. The energy of the charge-separated intermediate (protonated proline-deprotonated substrate) is calculated to be quite low, in accordance with measured pK(a) values. The results of the two models are used to evaluate the methodology employed in modeling enzyme active sites using quantum chemical cluster models.
|
|
| 4. |
- Sevastik, Robin, et al.
(author)
-
Reaction Mechanism of cis-3-Chloroacrylic Acid Dehalogenase : A Theoretical Study
- 2009
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:40, s. 9641-9649
-
Journal article (peer-reviewed)abstract
- The reaction mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is studied using the B3LYP density functional theory method. This enzyme catalyzes the hydrolytic dehalogenation of cis-3-chloroacrylic acid to yield malonate semialdehyde and HCl. The uncatalyzed reaction is first considered, and excellent agreement is found between the calculated barrier and the measured rate constant. The enzymatic reaction is then studied with an active site model consisting of 159 atoms. The results suggest an alternative mechanism for cis-CaaD catalysis and different roles for some active site residues in this mechanism.
|
|
| 5. |
|
|
