Recently, in Chemical Biology lecture, I learned about Kinetic Isotope Effects, where replacing hydrogen atoms with deuterium in a substrate can influence the rate of reaction.
The principle is that heavier atoms have a lower potential energy, so the energy required to access the transition state is greater. Thus the rate of reaction is quicker for the lighter atom. You can compare the rates of the deuterated reaction to the normal reaction and get a ratio that determines the nature of transitions at that position.
For example, if the ratio is greater than 2, then you know that the C-H bond breaks.
And if the ratio is between 1 and 1.5 or so, then you know that that carbon transitioned from sp3 to sp2 hyrbidization.
Finally, if the ratio is less than 1, then you know the opposite - that carbon went from sp2 to sp3.
Measurement of the r-Secondary Kinetic Isotope Effect for the Reaction Catalyzed by Mammalian Protein Farnesyltransferase
J. Pais, et al. JACS 2006
Anyway, this paper used the kinetic isotope effect to analyze the kinetics of farnesyltransferase, which is a pretty important enzyme required for helping lots of proteins associate with the cell membrane (including Ras!). Because product release is the limiting step for the reaction, they had to use low substrate concentration to mimic single turnover kinetics. So, they radiolabelled FPP and determined the extent of reaction at various time points. Because the observed KIE decreased with % of reaction, they had to extrapolate backward to approximate 0% of reaction.
They concluded from their data that C1 undergoes a dissociative transition state, which really doesn't mean anything to me....*shrug*. And the KIE was different for another peptide that they used, so I don't know what the deal is. Interestingly, Mg2+ speeds up the reaction, but doens't have an effect on the KIE, suggesting that the ion is not involved in the conformational rearrangement of FPP that they propose precedes the reaction.
Chlorination by a Long-Lived Intermediate in the Mechanism of Flavin-Dependent Halogenases
Yeh, Walsh, et al. Biochemistry 2007
This was an article by that guy here I really like. Basically, they characterize this enzyme RebH that creates 7-chlorotryptophan in the biosynthesis of rebeccamycin, an antitumor compound.
They initially thought that HOCl is generated at the active site via FAD, O2, and NaCl, and that the OCl- ion is what performs the chlorination. However, they demonstrate that the enzyme retains the chlorine on a lysine nitrogen, and uses that instead. They did this a number of ways, including making arguments that lysine blocks OCl- from channeling through the enzyme to the tryptophan substrate via a crystal structure they got, some radiolabelled HPLC reactions, and showing that OCl- could just float away if that was responsible, yet enzymatic activity remained for hours. I liked it - very clear and made sense.
The principle is that heavier atoms have a lower potential energy, so the energy required to access the transition state is greater. Thus the rate of reaction is quicker for the lighter atom. You can compare the rates of the deuterated reaction to the normal reaction and get a ratio that determines the nature of transitions at that position.
For example, if the ratio is greater than 2, then you know that the C-H bond breaks.
And if the ratio is between 1 and 1.5 or so, then you know that that carbon transitioned from sp3 to sp2 hyrbidization.
Finally, if the ratio is less than 1, then you know the opposite - that carbon went from sp2 to sp3.
Measurement of the r-Secondary Kinetic Isotope Effect for the Reaction Catalyzed by Mammalian Protein Farnesyltransferase
J. Pais, et al. JACS 2006
Anyway, this paper used the kinetic isotope effect to analyze the kinetics of farnesyltransferase, which is a pretty important enzyme required for helping lots of proteins associate with the cell membrane (including Ras!). Because product release is the limiting step for the reaction, they had to use low substrate concentration to mimic single turnover kinetics. So, they radiolabelled FPP and determined the extent of reaction at various time points. Because the observed KIE decreased with % of reaction, they had to extrapolate backward to approximate 0% of reaction.
They concluded from their data that C1 undergoes a dissociative transition state, which really doesn't mean anything to me....*shrug*. And the KIE was different for another peptide that they used, so I don't know what the deal is. Interestingly, Mg2+ speeds up the reaction, but doens't have an effect on the KIE, suggesting that the ion is not involved in the conformational rearrangement of FPP that they propose precedes the reaction.
Chlorination by a Long-Lived Intermediate in the Mechanism of Flavin-Dependent Halogenases
Yeh, Walsh, et al. Biochemistry 2007
This was an article by that guy here I really like. Basically, they characterize this enzyme RebH that creates 7-chlorotryptophan in the biosynthesis of rebeccamycin, an antitumor compound.
They initially thought that HOCl is generated at the active site via FAD, O2, and NaCl, and that the OCl- ion is what performs the chlorination. However, they demonstrate that the enzyme retains the chlorine on a lysine nitrogen, and uses that instead. They did this a number of ways, including making arguments that lysine blocks OCl- from channeling through the enzyme to the tryptophan substrate via a crystal structure they got, some radiolabelled HPLC reactions, and showing that OCl- could just float away if that was responsible, yet enzymatic activity remained for hours. I liked it - very clear and made sense.
And then, today on the front page of the New York Times.com, I read a summary of research that fed boys and men a diet of foods with high glycemic index and found a correlation with acne, presumably due to the triggered release of insulin, androgen, and other hormones that stimulate oil production.
Bacterially Speaking
Bassler/Losick Cell 2006
This was a cool review covering bacterial signaling. Some of this was review for me, after writing my review on Quorum Sensing in Staph. aureus last semester, but there are some pretty cool examples.
I knew about Vibrio fischerii, which produce bioluminescence in deep sea squids once they sense a high enough population through secreted acyl-homoserine lactones. Most of the gram-positive signals are peptides (including the Staph. cyclic AIPs or autoinducing peptides). Interestingly, there are some molecules, such as AI-2, that serve in cross-species communication.
Some molecules (quinolones from Pseudomonas) are hydrophobic, and so the bacteria will actually pinch off a portion of its membrane along with the molecule to encapsulate it and send it along. Amazingly, they can also package other types of molecules with it that serve to kill other types of bacteria that intercept the signal. Other bacteria secrete anti-quorum sensing molecules, such as proteases or lactonases that degrade signal, and we humans might even have a few circulating through the bloodstream (called paraoxonases, whose endogenous substrates still haven't been identified)! Other bacteria will just eat up the signal of neighbors (e.g. E. coli) to prevent them from communicating.
Many of these signals activate two-component systems, usually a histidine kinase that phosphorylates the aspartate of a response-regulator protein, that often comes in the form of a transcription factor, instituting development programs or other plans by influencing the expression of over >100 genes in many cases.
There are other examples - B. subtilis that are nutritionally deprived will start sporulation. About half of the population will not sporulate initially - these members will get targetted by a sporulation-induced killing system that basically kills these neighbors and uses them for food, until it's absolutely necessary to form spores.
There are more friendly examples of interspecies communication, including the Nod system we learned about in AP Bio where Rhizobium will grow on plant mycorrhizae that secrete the signal.
Nutritional Control of Elongation of DNA Replication by (p)ppGpp
Wang, et al. Cell 2007
I haven't read this paper yet, though I'm not sure I will get to it. In fact, I probably won't. But, according to the abstract, (p)ppGpp serves as a signal of nutrient deprivation that quickly and potently stops bacterial DNA replication by binding to primase. Awesome....I guess.
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