Difference between revisions of "Talk:Glycoside Hydrolase Family 134"

Revision as of 16:03, 30 November 2016

Dear CAZypedia curators,

GH134 enzymes are syn protonators: in the structure 5JUG in complex with mannopentaose, the proton donor Glu45 (Gln mutant) is clearly syn-positioned (near O5 of the mannose-ring in subsite -1), and the glycosidic bond is indeed out of the exo-anomeric effect. Maybe a short sentence on this can be included in this GH134 CAZypedia-entry?

With kindest regards, Wim

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 GH134 enzymes are syn protonators: in the structure 5JUG in complex with mannopentaose, the proton donor Glu45 (Gln mutant) is clearly syn-positioned (near O5 of the mannose-ring in subsite -1), and the glycosidic bond is indeed out of the exo-anomeric effect. Maybe a short sentence on this can be included in this GH134 CAZypedia-entry?
-The authors of the otherwise truly beautiful article with structures of a GH134 beta-mannanase (Jin et al, ACS Cent. Sci. 2016) have however missed something else that is essential about its mechanism. Please bear with me for the following, as I will eventually suggest only a small compromising corrective change for this CAZypedia-entry.
-The subsites -3 to +2 spanning mannopentaose complex 5JUG as well as the subsites -3 to -1 spanning mannotriose complex 5JU9 both show a surprising 1C4 inverted chair conformation for the mannose moiety occupying subsite -1. For the conformational reaction itinerary, the authors have correctly deduced that this implies that one of these inverted chairs must have been a 3S1 conformation, with the reaction proceeding through a transition state that is close to a 3H4 half-chair.
-The authors have however chosen to only consider that the central 1C4 mannose in the man5-complex is the productive start-conformation, thus that the observed 1C4 in the man3-complex was a 3S1 at the end of the substitution, leading to a 1C4-start to 3H4-like-TS to 3S1-end reaction itinerary, the latter collapsing to the 1C4 after the bond substitution is over. And only this route was further investigated with MD and QM/MM.
-The alternative 3S1-start to 3H4-like-TS to 1C4-end itinerary was not considered and no reason thereof was given. But especially the 1C4 in the man3-complex indicates that this is the actual itinerary.
-I have quickly drawn and minimized some ring-conformations of 1,4-dimethyl beta-mannoside:
-,4-dimethyl-beta mannoside; MMFF94s minimisation
-C1 401.4 kJ/mol = 95.6 kcal/mol
-C4 435.2 kJ/mol = 103.6 kcal/mol
-S1 466.1 kJ/mol = 111.0 kcal/mol
-S1 439.6 kJ/mol = 104.7 kcal/mol
-S1/1C4 -7.4 kcal/mol
-S1/4C1 -15.4 kcal/mol
-C4/4C1 -8.0 kcal/mol
-,4-dimethyl-beta mannoside; Amber-GAFF minimisation
-C1 29.7 kJ/mol = 7.1 kcal/mol
-C4 50.6 kJ/mol = 12.0 kcal/mol
-S1 74.5 kJ/mol = 17.7 kcal/mol
-S1 62.9 kJ/mol = 15.0 kcal/mol
-S1/1C4 -5.7 kcal/mol (*)
-S1/4C1 -10.6 kcal/mol
-C4/4C1 -4.9 kcal/mol
-Taking the lower estimate (*), the 1C4 inverted chair of a beta-mannoside is about 6 kcal lower in internal energy than the 3S1. In other words, a 1C4-start to 3H4-like-TS to 3S1-end reaction itinerary has a conformational energy burden of 6 kcal/mol more than a 3S1-start to 3H4-like-TS to 1C4-end itinerary has. The Hammond-postulate (see intro https://en.wikipedia.org/wiki/Hammond's_postulate ) then says that a 3H4-like-TS is not only closer in energy, but also closer in shape, to a 3S1 than it is to a 1C4. Applying this postulate to glycosidic bond substitutions gives the reason why one expects these to always proceed from a skew-start to half-chair-like-TS to chair-end.
-Moreover and surprisingly, the man3-complex also shows a 1C4 in subsite -1, although when subsite +1 is emptied after the reaction one would expect the -1 sugar to revert to its 4C1 ground state chair (as is often seen in other GH complexes). Apparently this enzyme has a -1 subsite that extra stabilizes a 1C4 inverted chair (calculated difference 1C4-4C1 is 5 kcal/mol, plus 2 kcal/mol more otherwise one would see double occupancies). The conformational energy burden (this alone, not even taking account the chemical reaction barrier) for going from the 1C4 to the 3H4-like-TS may then even surpass 10 kcal/mol for this enzyme, which is enormous in comparison to the by Yin et al calculated 17 kcal/mol total reaction free energy barrier. This then safely excludes a 1C4 as the start of the reaction itinerary.
-Extra stabilizing an inverted 1C4 chair in a 3S1-start to 3H4-like-TS to 1C4-end itinerary is advantageous because of the Bell-Evans-Polanyi principle (for intro see further in Wikipedia's Hammond postulate): this further decreases the reaction energy barrier and brings the 3S1-start even closer in shape to the 3H4-like-TS.
-And why does the man5-complex show a 1C4 inverted chair in the -1 subsite? Simply because it is lower in energy than the 3S1, thus most of the time an 1C4 is the conformation present there, but it is not the productive conformation. It is actually logical not to find a 3S1 there: the more a 3S1 would be stabilized by the enzyme, the higher the energy barrier for the reaction.
-What is truly remarkable and novel for the mechanism of this GH-enzyme is that an "equator to south pole" 3S1-start to 3H4-like-TS to 1C4-end itinerary (alternative-ALPH) is used for a substrate that has a scissile beta-equatorial glycosidic bond, and that it succeeds this by completely flipping the glycosidic ring. It is a real pity that the authors have missed this in an otherwise very beautiful article, and I wonder how that can be corrected.
-For the GH134 wikipedia entry, at "Kinetics and Mechanism", may I humbly suggest that its last sentence mentiones that because of the observed 1C4 inverted chair conformations in subsite -1, the itinerary likely proceeds through a 3H4-like transition state, which certainly remains a true statement. But please consider to remove the direction of the itinerary.
 With kindest regards,
 Wim

Difference between revisions of "Talk:Glycoside Hydrolase Family 134"

Revision as of 16:03, 30 November 2016

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