I think we are having a problem with the definition of "reversible" and "irreversible". By Lijun's definition the reaction is irreversible because it proceeds from far from equilibrium toward equilibrium. That situation is more a property of the system than the enzyme. If you make the enzyme 1000 times faster the reaction will proceed more quickly toward equilibrium despite the fact that the reverse reaction is also 1000 times faster. The reverse reaction doesn't matter when there is no product to act upon.
The original question was about having "a reversible epimerase and I want to mutate it into an inreversible(sic) one". Clearly the poster is talking about a property of the enzyme. I interpreted this question to be a request to differentially change the forward and backward reaction rates, but I could be mistaken. Maybe the original poster could clarify the question. Dale Tronrud On 05/21/10 13:53, Lijun Liu wrote: >> If I understand what you are saying, I think it is too. >> >> You imply that asymmetry in the enzyme results in two isomerase >> pathways. This may be true, but it has no consequence on the prospects >> for irreversibility. To avoid confusion, let's call these pathways "D" >> and "S". Both the "D" and "S" pathways would have their own kf and kr >> kinetic constants such that kf_D/kr_D = kr_S/kf_S = Keq, which >> reflects the dG of the reaction. When the dG is close to zero for the >> isomerase reaction (which I assume here), then you can't make it >> irreversible. > ＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝ > This is not the case, at least in part. Such kind of enzymes, if no > cofactor-needed, use the identical intermediate for the mirror symmetric > reaction. For the D <--> Intermediate <--> S reaction, the enzyme uses > the same pathway. Enzymes, for example, glutamate racemase and > aspartate racemase, use a kind of psudo-mirror symmetric alignment at > the active site to adapt the binding of D or S isomer in the half A.S., > respectively. Other 3 atoms associated to the chiral center keeps fixed > relative conformation during the inversion. > > Standard dG(0) of such a reaction is 0. However, at the time when > enzyme works (for example, cell needs D-ASP in an almost pure L-ASP > environment), the racemase moves L-Asp to D-Asp, in this regard, the dG > of the reaction (not standard) is not 0. > > Your last sentence means: for a reaction (assuming dG(0) = 0 like > racemic reaction) almost reaches EQ (dG ~ 0), you cannot make it > irreversible----this is true. Just please do not forget: such kind of > enzymes work when the D <--> S EQ is highly broken by nature (dG << 0) > [not dG(0)]. > > Hopefully I explained clearly! > > Lijun > > >> >> James >> >> >>> All natural epimerases, isomerase and racemases use a mechanism based >>> on L-amino acids to deal with a mirror-symmetric (quasi-, sometimes) >>> reaction. In another word, these enzymes use a non-mirror symmetric >>> structure to deal with a mirror-symmetric reaction, which itself >>> causes the asymmetric kinetics for different direction, though the dG >>> is 0. The Arrhenius Law k = A*exp(-dE/RT) should be understood like >>> this: a mutation's effect to dE will be symmetric as Dale pointed >>> out. However, the effects on A are asymmetric. A is related to >>> intramolecular diffusion, substrate- and product-binding affinity, >>> etc. That is why with mutation these enzymes changed their kinetics >>> on two directions differently. Please check glutamate racemase, >>> alanine racemase, aspartate racemase, DAPE epimerase, if you are >>> interested. Never a 1000 to 1000 relation! >>> >>> Thus, mutation is possible to make one direction more favored---the >>> point is you need the correct hit. Of course, such an experiment is >>> never a Maxwell's demon. >>> >>> Lijun >>> >>> >>> >>> >>> On May 19, 2010, at 8:51 AM, Maia Cherney wrote: >>> >>>> You absolutely right, I thought about it. >>>> >>>> Maia >>>> >>>> Marius Schmidt wrote: >>>>> Interestingly, Maxwell's demon pops up here, whoooo... , >>>>> don't do it. >>>>> >>>>> >>>>> >>>>> >>>>>> If you change the reaction rate in one direction 1000 times slower >>>>>> than >>>>>> in the other direction, then the reaction becomes practically >>>>>> irreversible. And the system might not be at equilibrium. >>>>>> >>>>>> Maia >>>>>> >>>>>> R. M. Garavito wrote: >>>>>> >>>>>>> Vinson, >>>>>>> >>>>>>> As Dale and Randy pointed out, you cannot change the ΔG of a >>>>>>> reaction >>>>>>> by mutation: enzyme, which is a catalyst, affects only the >>>>>>> activation >>>>>>> barrier (ΔE "double-dagger"). You can just make it a better (or >>>>>>> worse) catalyst which would allow the reaction to flow faster (or >>>>>>> slower) towards equilibrium. Nature solves this problem very >>>>>>> elegantly by taking a readily reversible enzyme, like an >>>>>>> epimerase or >>>>>>> isomerase, and coupling it to a much less reversible reaction which >>>>>>> removes product quickly. Hence, the mass action is only in one >>>>>>> direction. An example of such an arrangement is the triose >>>>>>> phosphate >>>>>>> isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) >>>>>>> reaction pair. TIM is readily reversible (DHA <=> G3P), but G3P is >>>>>>> rapidly converted to 1,3-diphosphoglycerate by GAPDH. The >>>>>>> oxidation >>>>>>> and phosphorylation reactions of GAPDH now make TIM "work" in one >>>>>>> direction. >>>>>>> >>>>>>> Since many epimerases are very optimized enzymes, why not consider >>>>>>> making a fusion with a second enzyme (like a reductase) to make the >>>>>>> system flow in one direction. Of course, this depends on what you >>>>>>> want to do with the product. >>>>>>> >>>>>>> Cheers, >>>>>>> >>>>>>> Michael >>>>>>> >>>>>>> /****************************************************************/ >>>>>>> /R. Michael Garavito, Ph.D./ >>>>>>> /Professor of Biochemistry & Molecular Biology/ >>>>>>> /513 Biochemistry Bldg. / >>>>>>> /Michigan State University / >>>>>>> /East Lansing, MI 48824-1319/ >>>>>>> /Office:// //(517) 355-9724 Lab: (517) 353-9125/ >>>>>>> /FAX: (517) 353-9334 Email: rmgarav...@gmail.com >>>>>>> <mailto:rmgarav...@gmail.com> >>>>>>> <mailto:garav...@gmail.com>/ >>>>>>> /****************************************************************/ >>>>>>> >>>>>>> >>>>>>> >>>>>>> On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: >>>>>>> >>>>>>> >>>>>>>> Hi, >>>>>>>> >>>>>>>> I'm more of a Fourier coefficient kind of guy, but I thought that a >>>>>>>> ΔG of zero simply corresponded to an equilibrium constant of >>>>>>>> one. You >>>>>>>> can certainly have reversible reactions with other equilibrium >>>>>>>> constants. >>>>>>>> In fact I think "irreversible" reactions are simply ones where the >>>>>>>> equilibrium constant is so far to one side that, in practice, the >>>>>>>> reaction >>>>>>>> always goes all the way to product. >>>>>>>> >>>>>>>> As Randy pointed out the enzyme cannot change the ΔG (or the >>>>>>>> equilibrium >>>>>>>> constant). You could drive a reaction out of equilibrium by >>>>>>>> coupling it >>>>>>>> to some other reaction which itself is way out of equilibrium >>>>>>>> (such as >>>>>>>> ATP hydrolysis in the cell) but I don't think that's a simple >>>>>>>> mutation of >>>>>>>> your enzyme. ;-) >>>>>>>> >>>>>>>> Dale Tronrud >>>>>>>> >>>>>>>> On 05/18/10 00:31, Vinson LIANG wrote: >>>>>>>> >>>>>>>>> Dear all, >>>>>>>>> >>>>>>>>> Sorry for this silly biochemistory question. Thing is that I >>>>>>>>> have a >>>>>>>>> reversible epimerase and I want to mutate it into an >>>>>>>>> inreversible one. >>>>>>>>> However, I have been told that the ΔG of a reversible >>>>>>>>> reaction is zero. >>>>>>>>> Which direction the reaction goes depends only on the >>>>>>>>> concentration of >>>>>>>>> the substrate. So the conclusion is, >>>>>>>>> >>>>>>>>> A: I can mutate the epimerase into an inreversible one. But it >>>>>>>>> has no >>>>>>>>> influence on the reaction direction, and hence it has little mean. >>>>>>>>> >>>>>>>>> B: There is no way to change a reversible epimerase into an >>>>>>>>> inversible one. >>>>>>>>> >>>>>>>>> Could somebody please give me some comment on the two conclution? >>>>>>>>> >>>>>>>>> Thank you all for your time. >>>>>>>>> >>>>>>>>> Best, >>>>>>>>> >>>>>>>>> Vinson >>>>>>>>> >>>>>>>>> >>>>>>>>> >>>>>>>>> >>>>> >>>>> Dr.habil. Marius Schmidt >>>>> Asst. Professor >>>>> University of Wisconsin-Milwaukee >>>>> Department of Physics Room 454 >>>>> 1900 E. Kenwood Blvd. >>>>> Milwaukee, WI 53211 >>>>> >>>>> phone: +1-414-229-4338 >>>>> email: m-schm...@uwm.edu <mailto:m-schm...@uwm.edu> >>>>> http://users.physik.tu-muenchen.de/marius/ >>>>> >>>>> >>>>> >>> >>> Lijun Liu >>> Cardiovascular Research Institute >>> University of California, San Francisco >>> 1700 4th Street, Box 2532 >>> San Francisco, CA 94158 >>> Phone: (415)514-2836 >>> >>> >>> >> > > Lijun Liu > Cardiovascular Research Institute > University of California, San Francisco > 1700 4th Street, Box 2532 > San Francisco, CA 94158 > Phone: (415)514-2836 > > >
