Dear Ilya,

Please have a quick look to a paper recently published on that topic (see the link).

On Figure 3,page 702 of this,paper, several correlations between specific  functional behaviors of polycyclic substrates with CYP1A enzymes were tested.The logP descriptor failed to establish a correlation. What was found more important is the size of the substrates. In this paper a corelation with structural channels is found of key importance in P450 enzyme function. Have a good reading!

Best regards,

Philippe

Article Access channels to the buried active site control substrate ...

Hellow, Ilya!

You asked an interesting question.  I think that the Koschland's theory of induced conformity is applicable also for the hydrophobic substrates of enzymes that make charged products.. But not in respect of binding a substrate, but in respect of releasing the product. In this case hydrophobic substrates interact with the active center from inside the membrane, but the formed charged product must be released via the hydrophilic  channel from the active center. Therefore, all mathematical models should work but, say "in the reverse".

Privet!

Sasha

Dear Philippe,

With all due respect, with terms that monooxygenase kinetics of chimeric mammalian CYPs in yeast lysate exemplify CYPs in mammalian microsomes, you’re metaphorically comparing apples to oranges. Since the late 80’s (especially while at NCI/NIH I dealt with baculovirus and vaccinia virus-expressed CYP1As), I’ve followed the data of your team reaching excellent progress in developing the above chimeras helping understand CYPs catalyze diverse reactions as key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. However, non-applicability of the enzyme kinetics of chimeric fungal model to enzyme kinetics of (ER)membrane-bound mammalian CYPs is summarized in your own words: “This result suggests that polycyclic substrates could be differentiated by [chimeric – I.T.] CYP1A enzymes on the basis of their size…, and not of their hydrophobicity”.

Just the opposite feature is typical for enzyme-kinetics of membrane-bound monooxygenases in the Kingdom of Animalia. Membrane phospholipids are established as essential catalysis-determining components of monooxygenase activities of CYPs, both microsomal and purified (where phosphatidylcholines were used). Tons of publications verified the matter of the fact, among those there were our data on protonophores revealing two phospholipid-dependent hydrophobic zones in microsomal membrane specific for CYP1a1 and CYP1a2, then called “aniline hydroxylase” (Tsyrlov et al., Biochem. J. 160:75-83, 1976), and direct comparison of enzyme-kinetic parameters for microsomal, purified and the recombinant murine CYP1As (Tsyrlov et al., Ach. Biochem. Biophys. 307:259-266, 1993).

Moreover, a fellow of mine since the 80’s Pavel Anzenbacher and colleagues recently published a paper epitomizing the role for substrate hydrophobicity and ER phospholipids: “Catalytic domains of all studied CYPs were found to be partially immersed in the lipid bilayer, whereas the N-terminal part and F′/G′ loop are deeply immersed. The proximal side of the enzyme faces the cytosol, whereas the distal side, where openings of substrate access and product release channels to the active site are primarily located, points toward the lipid bilayer. Access channels with openings in the vicinity of the B/C and F/G loops are typically positioned below the lipid head groups, whereas the solvent channel points toward the membrane–water interface. This may indicate that membrane-anchored CYPs have evolutionarily adapted to facilitate uptake of nonpolar substrates from the membrane and uptake/release of polar substrates or products from/to the membrane–water interface” (Berka et al., J. Phys. Chem. 117:11556-11564, 2013).  

Best regards,

Ilya

Dear Sasha,

Glad to hear from you, and beg your pardon for delayed reply. First, I’d responded to my French colleague who, like many others working with chimerical and otherwise recombinant P450s, just overlooked (or simply ignored) that enzyme kinetics of mammalian membrane-anchored monooxygenases drastically dependent on surrounding phospholipids. Resulting from that, the Km, Ks, Kcat values all were several orders different from those of microsomal ones (i.e. non-physiological values, which scientific input was close to zero (по-русски говоря, ситуация c химерами описывается известной присказкой: «а вот еще картиночка, достойная пера – невинную сардиночку е@ут два осетра»).

Your thoughts regarding «hydrophobic substrates interact with the active center from inside the membrane” already require usage of the Hansch coefficient illuminating substrate distribution between two phases characterizing by different hydrophobicity. As for “product must be released via the hydrophilic channel from the active center”, and that “the models should work but, say "in the reverse", - it has some sense, and might be further elaborated.

What I meant while relating membraneous P450s to Koshland theory was based on the data in the beneath paper* published in 1979 in «Kinetics and Catalysis» (inasmuch as it's solid Russian journal1979, - it's pity we lazy idiots did not published the data internationally). There we used three substrates and inducers of P450, - and came to suggestion that inducer used the plasticity of P450 to establish enzyme new active centre configugation, which was «memorized» by means of membrane stuctures, So, the metabolic specificity of induced enzyme (i.e. specific position in the molecule undergoing monooxygenation) to many other substates started resembling that of the inducer itself.

What’s up?

Ilya

*Effect of Substrate and Inducer Structure on Catalytic Activity of Microsomal Monooxygenases. A. Anshits, V. Sokolovsky, S. Burilin, Ilya Tsyrlov. Kinetics and Catalysis 20:865-870, 1979)

Привет, Илья!

How nice to hear from an Old Friend!. Strange, but in my work, particulalrly when I was writing my book " Practical mitochondriology" (actually two books because for the Russian readers you have to write differently) I have encountered the same problems. The old paradigms, which we have studied in our Youth (in those cold winter days in old building, when we warmed ourselves with hot strong coffee with C2H5OH. Currently you have to consider events with not just one enzyme, but with functional complexes and finding the point of irreversibility.

Thank you for the reference. I agree, there are so many things that we made and not published. Sometimes, we were ahead of everybody for decades.

Good luck. Best regards to Irina.

Sincerely,

Sasha.