Hello,
I work with L-lysine monooxygenase, FAD is the co-factor and the enzyme is NAD(P)H dependent!
one of the mutations I am studying, is oriented very close to FAD isoalloxazine moiety. it is glutamate that was mutated to glutamine!
the mutant shows higher activity than the wild type, the difference I have during the anaerobic reduction with NADPH that I have 3 phases compared to 2 phases in the WT.
the second and third phases are identical to the WT in terms of Kcat and Km with NADPH variation.
the only difference is the first phase with higher both Kcat = 6 s-1 and km = 1 mM NADPH; compared to 1 s-1 and 0.2 for the WT, respectively.
the flavin spectra shows higher intensity broad peak in the wavelength range upon binding of the enzyme with NADPH!
there is no other spectral change at higher wavelengths that could be considered as a formation of charge-transfer complex!
the only explaination that I have right now is that NADPH binding unfavorable in the very early stages affecting the FAD environment and causing these spectral changes!
I appreciate if I can get more idea about that!
Thank you
This is by no means an inclusive answer, just a possibility. The new first phase could reflect a hydrogen bond making and/or breaking event that only occurs in the Gln mutant. Policycyclic aromatic cofactor produce strong "ring currents" that are sensitive reporters of perturbations of the local environment. A change in hydrogen bonding resulting in increased access to conformational states (increased flexibility) could account for broadening of the UV spectrum of the new (temporally first) state. Does the UV spectrum change in deuterium-enriched vs normal water? Other spectroscopic methods, particularly NMR and IR combined with isotope enriched Gln/oGlu may provide detailed information. In sumnary, the Glu to Gln mutation likely provides access to a new state (set of conformations) at innassessbly high free energy In the wild type enzyme. There may be a different reasons for your own observation. The only exception I'm suggesting is quite plausible given that small structural changes can yield large even delocalized changes in dynamics and accessible states.
I think that before expending too much effort on hypotheses, it's worth clarifying what you've observed.
First, I think you're pondering the results from reductive half-reaction experiments, not steady-state kinetics, right? And so these are not kcat and Km values; presumably, you're alluding to the concentration dependencies of observed rate constants?
Second, the spectra you show don't seem to be from an experiment that could really address the issues. You don't state concentrations and conditions, but the isosbestic around 330 nm and low absorbance in general below ~400 nm, and the absorbance of ~0.22 at 450 nm, suggest that you're showing the reaction of ~0.02 mM NADPH with ~0.02 mM enzyme. However, the concentration of NADPH is way below saturation, from what I glean from your description of the kinetic analysis. This is way too low to get spectra of intermediates. To maximize kinetic resolution, you need to saturate in NADPH. Sure, you'll obliterate the spectrum below ~400 nm, but that's the price you need to pay if to build up appreciable amounts of an intermediate. Only then should you pass judgement on whether there are charge-transfer complexes. But once you post such spectra, let's rethink!
Thank you for your comments Dr. Keith Constantine and Dr. Bruce Allan Palfey, I have never look to many of the strategy that you are suggesting to further investigate that, but I will try to do that.
Dr. Bruce Allan,
I have other spectra at higher NADPH concentration (saturation level) and like you said I won't be able to observe any change below 400 nm because of the excess NADPH I have.
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