Registration Dossier

Administrative data

Endpoint:
biochemical or cellular interactions
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Optimizing the Michaelis complex of trimethylamine dehydrogenase: identification of interactions that perturb the ionization of substrate and facilitate catalysis with trimethylamine base
Author:
Basran, J., Sutcliffe, M. J. and Scrutton, N. S.
Year:
2001
Bibliographic source:
J. Biol. Chem., 276(46), 42887-42892, 2001

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
study of interactions between TMA and the enzyme trimethylamine dehydrogenase
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Trimethyl-d9-amine HCl (99.7% D; chemical purity > 99 % determined by high performance liquid chromatography, NMR, and gas chromatography) was from CK Gas Products Ltd.
Specific details on test material used for the study:
no further details given

Test animals

Species:
other: not applicable (in vitro investigation)
Details on test animals and environmental conditions:
not applicable

Administration / exposure

Route of administration:
other: not applicable
Details on exposure:
not applicable
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
not applicable
Frequency of treatment:
not applicable
Post exposure period:
not applicable
Doses / concentrations
No. of animals per sex per dose:
not applicable
Control animals:
other: not applicable
Details on study design:
please refer to 'Any other information on materials and methods incl. tables'

Results and discussion

Details on results:
It is indicated that trimethylamine base (and not, as previously thought, the protonated cation) is the catalytically relevant form of the substrate in the ES complex. Tyr-60 and His-172 are the main residues responsible for stabilizing trimethylamine base in the ES complex. TMADH is poised to initiate flavin reduction by nucleophilic addition of the substrate lone pair at the flavin C4a atom by perturbing the substrate ionization by over 3 pH units (equivalent to 2 kJ/mol) on forming the ES complex.

Applicant's summary and conclusion

Conclusions:
The analyses of the reductive halfreaction of mutant forms of TMADH indicate that the trimethylamine base (and not, as previously thought, the protonated cation) is the catalytically relevant form of the substrate in the ES complex. The mutagenesis studies have identified Tyr-60 and His-172 as being the main residues responsible for stabilizing trimethylamine base in the ES complex. A role for residue Tyr-174 in the pH-dependent behavior of TMADH has also been ruled out. This work demonstrates that TMADH is poised to initiate flavin reduction by nucleophilic addition of the substrate lone pair at the flavin C4a atom by perturbing the substrate ionization by over 3 pH units (equivalent to 2 kJ/mol) on forming the ES complex. These studies are consistent with a mechanism of flavin reduction proposed in the authors previous work, and may have general implications for the mechanism of amine oxidation by flavoproteins.
Executive summary:

Recent evidence from isotope studies supports the view that catalysis by trimethylamine dehydrogenase (TMADH) proceeds from a Michaelis complex involving trimethylamine base and not, as thought previously, trimethylammonium cation. In native TMADH reduction of the flavin by substrate (perdeuterated trimethylamine) is influenced by two ionizations in the Michaelis complex with pKa values of 6.5 and 8.4; maximal activity is realized in the alkaline region. The latter ionization has been attributed to residue His-172 and, more recently, the former to the ionization of substrate itself. In the Michaelis complex, the ionization of substrate (pKa ~ 6.5 for perdeuterated substrate) is perturbed by ~ -3.3 to 3.6 pH units compared with that of free trimethylamine (pKa = 9.8) and free perdeuterated trimethylamine (pKa = 10.1), respectively, thus stabilizing trimethylamine base by ~ 2 kJ/mol. It isshown, by targeted mutagenesis and stoppedflow studies that this reduction of the pKa is a consequence of electronic interaction with residues Tyr-60 and His-172, thus these two residues are key for optimizing catalysis in the physiological pH range. It is also shown that residue Tyr-174, the remaining ionizable group in the active site that was not targeted previously by mutagenesis, is not implicated in the pH dependence of flavin reduction. Formation of a Michaelis complex with trimethylamine base is consistent with a mechanism of amine oxidation that was analysed in the authors previous computational and kinetic studies which involves nucleophilic attack by the substrate nitrogen atom on the electrophilic C4a atom of the flavin isoalloxazine ring. Stabilization of trimethylamine base in the Michaelis complex over that in free solution is key to optimizing catalysis at physiological pH in TMADH, and may be of general importance in the mechanism of other amine dehydrogenases that require the unprotonated form of the substrate for catalysis.