Characterization of the Transition State for the Hydride Transfer in a Model of the Flavoprotein Reductase Class of Enzymes

Mestres, Jordi; Duran, Miquel; Bertrán, Juan

doi:10.1006/bioo.1996.0008

Cited by 15 publications

(16 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In accord with this, studies of model dihydropyridine hydride transfers indicate that significant charge develops in the transition state (Kurz & Frieden, 1975). Furthermore, a semiempirical quantum mechanical study of hydride transfer from dihydronicotinamide to isoalloxazine (Mestres et al, 1996) found, using either the AM1 or PM3 Hamiltonians, that the flavin in the transition state develops a charge of about -0.5. Therefore, it is reasonable to expect the active site electrostatic field to provide significant stabilization of the partial charge of the transition state, but this is not our experimental finding with PHBH.…”

Section: Discussionmentioning

confidence: 82%

Electrostatic Effects on Substrate Activation in para-Hydroxybenzoate Hydroxylase: Studies of the Mutant Lysine 297 Methionine

et al. 1997

View full text Add to dashboard Cite

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein monooxygenase that catalyzes the incorporation of one atom of molecular oxygen into p-hydroxybenzoate to form 3,4-dihydroxybenzoate. The enzyme activates the substrate at the 3 position to electrophilic substitution by lowering the pKa of the phenolic oxygen. The results presented here indicate that regions of positive potential in the active site facilitate this substrate activation, which is necessary for rapid hydroxylation. We have neutralized a positive point charge by mutating lysine 297 to methionine (K297M). This mutation changes an amino acid near the active site, but not directly in contact with the flavin or the substrate. A variety of transient state kinetic and static parameters have been determined with two substrates. The results indicate that the K297M mutant does not activate the substrate through phenolic ionization to the same extent as wild-type (WT) and yet remains a competent hydroxylase. However, catalysis by the mutant is slow compared to that of WT, particularly in the oxidative half-reaction. Thus, normally quite labile oxygenated flavin intermediates encountered in the hydroxylation pathway of WT p-hydroxybenzoate hydroxylase are stabilized and their decay is rate limiting in the K297M turnover. Electrostatic potential calculations offer an explanation for the lack of substrate activation. The stability of the oxidative reaction intermediates seems to be related to a lower degree of substrate activation.

show abstract

Section: Discussionmentioning

confidence: 82%

Electrostatic Effects on Substrate Activation in para-Hydroxybenzoate Hydroxylase: Studies of the Mutant Lysine 297 Methionine

et al. 1997

View full text Add to dashboard Cite

show abstract

“…On the other hand, Bertrán et al. , have carried out an extensive analysis of the hydride transfer in different model systems in terms of valence bond structures to understanding the coupling between the hydrogen motion and the electronic shift along the reaction pathway for HT step …”

Section: Resultsmentioning

confidence: 99%

On Transition Structures for Hydride Transfer Step in Enzyme Catalysis. A Comparative Study on Models of Glutathione Reductase Derived from Semiempirical, HF, and DFT Methods

et al. 1996

View full text Add to dashboard Cite

As a model of the chemical reactions that take place in the active site of gluthatione reductase, the nature of the molecular mechanism for the hydride transfer step has been characterized by means of accurate quantum chemical characterizations of transition structures. The calculations have been carried out with analytical gradients at AM1 and PM3 semiempirical procedures, ab initio at HF level with 3-21G, 4-31G, 6-31G, and 6-31G basis sets and BP86 and BLYP as density functional methods. The results of this study suggest that the endo relative orientation on the substrate imposed by the active site is optimal in polarizing the C4-Ht bond and situating the system in the neighborhood of the quadratic region of the transition structure associated to the hydride transfer step on potential energy surface. The endo arrangement of the transition structure results in optimal frontier HOMO orbital interaction between NADH and FAD partners. The geometries of the transition structures and the corresponding transition vectors, that contain the fundamental information relating reactive fluctuation patterns, are model independent and weakly dependent on the level of theory used to determine them. A comparison between simple and complex molecular models shows that there is a minimal set of coordinates describing the essentials of hydride transfer step. The analysis of transition vector components suggests that the primary and secondary kinetic isotope effects can be strongly coupled, and this prompted the calculation of deuterium and tritium primary, secondary, and primary and secondary kinetic isotope effects. The results obtained agree well with experimental data and demonstrate this coupling.

show abstract

“…Quantum chemical calculations on coenzymes and other enzyme ligands, in isolation or interacting with a limited number of atoms, have provided additional insight into their function and properties during enzyme catalysis 20–24. Recently, quantum chemistry has been used to elucidate the precise events occurring at the active site of ribonuclease A 25.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of NAD(P)H:Quinone reductase: Ab initio studies of reduced flavin

Cavelier

Amzel

2001

Proteins

View full text Add to dashboard Cite

NAD(P)H:quinone oxidoreductase type 1 (QR1, NQO1, formerly DT-diaphorase; EC 1.6.99.2) is an FAD-containing enzyme that catalyzes the nicotinamide nucleotide-dependent reduction of quinones, quinoneimines, azo dyes, and nitro groups. Animal cells are protected by QR1 from the toxic and neoplastic effects of quinones and other electrophiles. Alternatively, in tumor cells QR can activate a number of cancer chemotherapeutic agents such as mitomycins and aziridylbenzoquinones. Thus, the same enzyme that protects the organism from the deleterious effects of quinones can activate cytotoxic chemotherapeutic prodrugs and cause cancer cell death. The catalytic mechanism of QR includes an important initial step in which FAD is reduced by NAD(P)H. The unfavorable charge separation that results must be stabilized by the protein. The details of this charge stabilization step are inaccessible to easy experimental verification but can be studied by quantum chemistry methods. Here we report ab initio quantum mechanical calculations in and around the active site of the enzyme that provide information about the fine details of the contribution of the protein to the stabilization of the reduced flavin. The results show that (1) protein interactions provide approximately 2 kcal/mol to stabilize the planar conformation of the reduced flavin isoalloxazine ring observed in the X-ray structure; (2) the charge separation present in the reduced planar form of the flavin is stabilized by interactions with groups of the protein; (3) even after stabilization, the reduction potential of the cofactor remains more negative than that of the free flavin, making it a better reductant for a larger variety of quinones; and (4) the more negative reduction potential may also result in faster kinetics for the quinone reduction step.

show abstract

Characterization of the Transition State for the Hydride Transfer in a Model of the Flavoprotein Reductase Class of Enzymes

Cited by 15 publications

References 12 publications

Electrostatic Effects on Substrate Activation in para-Hydroxybenzoate Hydroxylase: Studies of the Mutant Lysine 297 Methionine

Electrostatic Effects on Substrate Activation in para-Hydroxybenzoate Hydroxylase: Studies of the Mutant Lysine 297 Methionine

On Transition Structures for Hydride Transfer Step in Enzyme Catalysis. A Comparative Study on Models of Glutathione Reductase Derived from Semiempirical, HF, and DFT Methods

Mechanism of NAD(P)H:Quinone reductase: Ab initio studies of reduced flavin

Contact Info

Product

Resources

About