Enzyme Kinetics

I've taken two biochemistry courses now, structural biochem and metabolic biochem. I guess this is a pretty standard undergrad curriculum. Both dealt with enzyme kinetics fairly extensively, and I realized today, sitting in class and going over bi bi multi-substrate mechanisms--ping pong and ordered sequential--that both classes took a totally different approach in allowing us to think about enzyme kinetics.

In structural biochem took an approach where we did Michaelis-Menton, inhibition, etc. but it was always in a "how can we change this interaction in the active site with the substrate to affect catalysis". It was pretty concrete to me, because you can mess with concrete, chemical interactions, like a hydrogen bonding interaction between residues, or some residue that kinks the protein out of alignment conformationally. Km and kcat had a directly linked cause--and that cause was intermolecular interactions.

In metabolic, it's sort of at this gross scale of when the substrate binds and when the substrate comes off. It's taking a step back from structure, and thinking about timing and mechanism at a larger scale. It's no longer this glutamate residue--it's just substrate A binds before substrate B. The mathematics feels so abstract, because I feel like I can't really picture in my head the fact that the blobular enzyme is undergoing small scale intermolecular interactions with the blobular substrate. It's just line drawings; it's schematics. I like it ok, and it's certainly a powerful approach. But what draws me to chemistry is structure, reactivity, and function. It's this viceral visual way of thinking about molecules coming together in space. And that's kind of lacking for me in Lineweaver-Burke plot after Lineweaver-Burke plot.

Incidentally, in reading the enzyme kinetics page on Wikipedia, I found it ammusing that "bi bi ping pong" was a totally technical term, and not a Ron-ism. Ron throws in conventional technical terms (which are, to be fair, often goofy sounding) with his own enzyme kinetics lingo, so sometimes hard to tell what is a general term that the biochemical community uses, and what terms are unique to him and his research. For example, he studies an enzyme that never lets go of its substrate, and so he likes to call those enzymes "Promethian enzymes". If you recall, Promethius in Greek mythology stole fire from Zeus, and to be punished, he had to be chained to a rock for the rest of his eternity while eagles ate out his liver. Then he would re-grow a liver and the cycle would repeat. So I guess that is an apt analogy.

How I'm spending my weekend:

Pictures tell this story better than words

pKas

One thing I am apparently good at:

I am a pKa machine. I basically can tell you the pKa of just about any organic molecule off the top of my head. If I don't know it, I can usually extrapolate what it is from related structures and by how well you can stabilize the conjugate base. I continually impress people by this ability and by how rarely I need a pKa table.

Usually I have a shoddy memory for rote memorization, so I have no idea how this stuck so well.

Summer in Germany; Polyketide Biosynthesis

As I may or may not have posted before, this summer I'm going to do a lab internship in Jena Germany at the Hans Knoll Institute: Leibniz Institute for Natural Products Research and Infection Biology in the department of biomolecular chemistry in Christian Hertweck's group.

The story of how this works is the following: I was writing a paper for my biochemistry seminar which focused on chemical biology and I stumbled upon work he was doing with polyketide biosynthetic enzymes in the aureothin (a Streptomyces thioluteus polyketide) pathway. Specifically, AurH, an enzyme catalyzes the chiral oxidation of a THF ring. This paper caught my attention as a mechanistic paper that dissected how the enzyme worked to act as a catalyst and this paper* used the enzyme in a stereospecific total chemoenzymatic synthesis. I read about how they figured which enzyme did the oxidation using molecular biology methods, along with how they figured out how the enzyme worked with biochemical methods, and then their application synthetically. It intrigued me because it was basically exactly the sort of research I want to be doing. It seamlessly integrates biology and chemistry. It relies on both synthetic organic chemistry and molecular biology. Dissecting mechanistically interesting biosynthetic steps that are difficult to mimic with classical reagents is just a really fascinating area of study to me. So I wrote a review paper for my class about research from his lab.

So one day, kind of on a whim I emailed Christian Hertweck, the PI with my CV. He was impressed with my background and my strong desire to do interdisciplinary science and offered me a summer internship at the HKI. I've been in correspondance all year working out the details. So I will be in Germany this summer doing chemical biology research.

It hasn't really hit me that I will be in Germany this summer doing exactly the sort of science I want to be doing. It seems like such a lucky shot in the dark.

I'm so pumped for this summer and next year. I like learning, so classes are alright, but what I really like is doing research. It reminds me "oh yes, this is why I study science."

Also, this will be my fourth consecutive summer in a lab, along with doing research in my prof's lab during the year for a bit and an undergraduate thesis. Although not my motivation for wanting to be in the lab in the summer, that's gotta look attractive to graduate schools, right?

*As a tangent, I also really like the journal ChemBioChem, which is a European chemical biology journal published by Wiley. ACS Chemical Biology puts out a few interesting articles, but it feels uninspiring a lot of the time (and virtually indistinguishable from what goes into ACS Biochemistry), and Nature Chemical Biology seems differently focused. There is some interesting bioorganic work being done here but the the mentality seems different somehow. It seems like the organic community is more seemlessly integrated in this European journal. More on this later.

Last year of college?

My course schedule for next year (my last year of college, eek) looks tentatively like this:

First semester:
--Thesis
--Analytical chemistry; graduation requirement. Enough said. I don't think anyone enjoys propagating error and if they do they are a freak of nature.
--Imperial Christianity; religion class, gotta get those liberal arts requirements done, plus it's with a badass prof
--Topics in Biochemistry; a mellow seminar that meets once a week for an hour and a half to discuss papers, next year the topic is the role of metals in biochemistry

Second semester:
--Thesis
--Cellular biology; only going to take the lecture portion of this, though. I've had experience culturing cells and doing Westerns. The only thing I feel like I'm really missing is learning how to use the qPCR machine. In any case I don't want to be in cell bio lab when doing a thesis, but I really would like an introduction to the field beyond what I've picked up over the past few years (i.e. run away from the MAP kinase pathway).
--Advanced inorganic chemistry; it's a half-unit lecture only course. I-chem is just starting to pick up for me since we are doing transition metals and you get to go into the really cool stuff in there; it also derives group theory.
--Intro drama; an English class, I feel like I could use some more literature classes in my life

I have enough credits that I could drop either cell or advanced i-chem second semester and still be good to graduate and may very well do that so I can spend more time on my thesis. More importantly, my classes are clumped together well so that I have a lot of time to spend in the lab.

Yesterday my boyfriend was sitting in the chemistry lounge, listening to a podcast of an NPR interview with David Foster Wallace. One of my chemistry professors walks by and starts chatting about how David Foster Wallace taught at Pomona at the same time he taught at Pomona.

Alan (my professor): So apparently he wasn't a very happy guy.
My boyfriend: Well, you know, that's what writing about the postmodern condition will do to you.
Me: Or thinking about molecular orbital theory too much.
Alan: Oh that was sharp. Is this a disgruntled chem 324 student I hear? [i.e. advanced mechanistic organic chemistry]

Yes, Alan. Yes it is. I have such a love hate relationship with MO theory. On the one hand it explains...just about everything more or less and is really fundamentally important for understanding chemistry. I switch between thinking the quantum mechanical explanations behind it are fascinating at a conceptual level and not particularly interesting in its details. Bond models feel like bullshit to me; with all the different iterations of orbital theory, I don't know how someone can graduate with a degree in chemistry thinking that it's any more than an amalgam of useful models that sort of kind of approximate reality and happen to explain shit well.

Usually I'm ok with this fact. If I felt that science was free of "woo woo" (as one of my bio profs liked to call things like lit theory and epistimology) then I would really be a headcase. At the same time I'm sick of hearing soc majors after taking a sociology of science class tell me that science is just socially constructed and no more legitimate than any other dicipline of study because, yeah, bonding models do--at some level--contain some "bullshit" but people have still used them to, like, make an LED out of a fucking semiconductor. And band theory is hella useful for explaining that behavior.

Stat therm

Last night my boyfriend asked me (he's a chemistry major, although a sophomore taking organic right now) what the reagent NBS did. I could answer him immediately and even describe the structure of the molecule and that NBS stood for "n-bromo succinamide".

Organic reagents, pKas, structures of the amino acids, NMR shifts and typical J-values, IR shifts, even some boiling points, this is all at the tip of my fingers. It's just stored in my head--anything organic or bioorganic is just there. I haven't forgotten it.

There's an incredible amount of information pertaining to organic chemistry and biochemistry that is just completely accessible in my head. I do not understand then, why it is so difficult to download thermodynamics equations in there. It's not like I enjoy memorizing pKas anymore than I enjoy memorizing thermodynamic definitions.

I realized earlier today that I have been approaching physical chemistry all wrong. I have a really shitty memory for information without context and so while some people can just cram equations into their head for tests that tends to not work for me. Organic rarely required memorization beyond a few weirdo reagents whose mechanisms were beyond the scope of the class; it was just mastering a few trends and then applying problem solving. Math is like this too, and physical chemistry is math. I need to learn the definitions and work from there and apply problem solving to derive everything I need.

Since I am uncomfortable with math as a language given my poor schooling in calculus, I have generally considered myself not mathematically facile enough to apply this approach to stat therm. I realized today, though, that the memorize special situations equations approach and memorizing derivations approach isn't going to work either, because there is just too much goddamn material for that. And I am facile enough with algebra and calculus...I just need to be confident that is the case. I need to work with the language and do the problem solving.

Why won't the algebra work out?

Fugacity is currently the bane of my existence.

Electron spin existential crisis

Last night while studying for an inorganic chemistry exam I have tomorrow, I suddenly had a huge existential crisis about not understanding magnetism.

It went something like this: how does MO theory tell us that oxygen should be paramagnetic and carbon should be diamagnetc?

Well, oxygen has unpaired electrons in its HOMO. Carbon has paired electrons in its HOMO. One or more unpaired electron leads to paramagnetism.

Okay, hold up. So why does oxygen want to have its electrons have the same spin when one goes into each degenerate orbital? Why, well, electrons repulse each other since they are negatively charged, thus if they can, they will go into separate degenerate orbitals (following Hund's rule). Due to the Pauli exclusion principle that means that electrons cannot have the same spin and position. If they have the same spin, they necessarily have different positions, which leads to less electrostatic repulsion. This is my qualitative understanding of the issue, and it is also--to my understanding--responsible for the phenomenon of exchange energy in half filled shells.

(Filling out orbital diagrams in this order has become intuitive to me after three years of undergraduate chemistry, but I guess I rarely step back and re-rationalize why it's the case).

So coming back to paramagnetism. This means that if those two electrons have the same spin, there is a permanent magnetic moment in the molecule (right? but what what is a magnetic moment anyway?!?! crap.) which means that it creates its own magnetic field. I recall the Lorentz Force law from physics (is this even exactly related?) although much of what I remember from electricity and magnetism involves a lot of fussing around with the right hand rule and being confused by which direction my thumb went. Anyway, if there is a permanent magnetic moment when put in an external magnetic field, the magnetic field can be parallel and thus be attracted. Whereas when a diamagnetic species is put in a magnetic field, since there are two electrons going in opposite directions, one of the vectors must necessarily oppose the magnetic field.

Okay, this makes sense, although I'm sure I butchered a few of the details. Then there's something something angular momentum vector something something spin momentum vector something something more quantum mechanics something something. But then I ask myself what is spin really? Why does it run parallel to the magnetic field. Why are parallel magnetic fields attracted to one another? Does it just have to do with the orientation of the electrons compared to the nucleus? WTF? Wait, wait, this was all beginning to make sense, and now it's all falling apart again.

So I waste hours of time on Wikipedia physics pages being confused.

It's funny that we use the same language as people who actually have a deeper level of understanding of these phenomena, but don't actually understand it at a deep level at all. It's all about shorthands to understand the parts we need to understand--in this case--for chemical reactivity and understanding MO theory.

The more chemistry I learn, the more of a head-case I become.

Junior Qualifying Examinations

So at Reed you have to pass a junior qualifying examination before you can register for your senior thesis. As an interdisciplinary major, I had to take both biology and chemistry. Biology was an open note/book/internet exam basically based on my coursework. I wrote an essay about haploinsufficiency in Marfan's syndrome fibrillin-1 gene and the elastin gene in Williams' syndrome and another essay where I wrote about the growth hormone axis and how nutrition (both being malnourished and obese) affected this axis. I also did some quantitative questions like calculating Tms of DNA strands and making buffers. I should hear back about it soon, although I'm afraid I might have gotten a conditional pass from messing up calculating TRIS on that buffer question, eeek. It's not a big deal if that happens, I just need to fix what I got wrong.

The chemistry qual was an oral exam where I was given this paper and had to learn as much as I could about it in 48 hours. Then they asked me questions, specifically about the paper, but really probing my organic chemistry knowledge as a whole. I had two options, an organic option and a biochem option, and I decided to go with the organic option. The biochem option was interesting too, it was on nicotine binding in the brain and how it is different from its inhibitory effects on your muscles (if it did hit the receptor in your muscles you'd be dead). But ultimately I felt more comfortable with the sort of questions they would ask in an organic qual, especially since my advanced synthetic final last semester was in the exact same format. Luckily, my organic paper was a synthetic paper and not a mechanistic paper--because for some reason the long lists of data tables with numbered compounds in mechanistic organic papers followed by colums of de's and ee's result in my eyes glazing over a lot of the time.

I think it overall went well. A lot of the questions were definitional--what is the kinetic enolate vs. thermodynamic enolate, what is an allene functional group, what is "latent stereochemistry". The main places I got tripped up on were mechanisms. I had this divinyl oxy-anion cyclopropyl Cope rearrangement mediated by a Brook rearrangement, and I started off drawing the reaction in a different perspective than I had in my notes which kind of threw me. The paper went to great pains to discuss why this enolate attacked from the more sterically hindered side (because it was reversible and resulted in the more stable transition state for the subsequent Cope) but to demonstrate it only showed what didn't happen due to steric repulsions--so there was no picture to look at what did happen. Which I knew, but it's just sort of difficult to pull all of that together visually on the board with weird bicyclic systems and the boat transition state and so forth. It went ok, I just had a moment where I was like "wait Connie..."

The other place I got tripped up due to nerves was the mechanism for oxidizing an enolate with a Davis oxaziridine. Again, it was ultimately ok, just a little bit of nerves. Also drawing 7 and 8 membered rings on the whiteboard was tricky!

The spectroscopy portion was easy since they had already assigned the peaks in my paper and my profs didn't ask me anything too difficult. Just like, why methyl groups attached to silyl groups were more upfield than methyls attached to carbons and whether this one set of diastereotopic methylnes were chemically equivalent or not, and why they had different J-values coupling to a neighboring proton that sort of thing.

It also struck me last night that wow, college has crammed a lot of chemistry into my head in the past 2 years.

So I should be hearing back about whether I passed, conditionally passed, or failed those shortly.

Nature Chemistry and Twitter

This month is the first issue of Nature Chemistry, and as far as I can tell they appear to have some sort of free preview for the first issue because I'm positive that Reed doesn't have access to this journal, but I've been able to access the full text PDFs. It seems like a good read, although I haven't had a chance to look at it all that thoroughly.

In addition, I have learned that all the major journals AND sigma aldrich have twitter feeds. As if I really needed to know about the latest advance in chiral chramotography in a two line twitter blurb, heh. It's sort of an interesting form of information flow, because I've noticed that this sort of abstract of an abstract catches my attention occasionally in ways that my RSS feeder doesn't.

I also learned that Nature Protocols gives out the occasional free featured protocol. This week it is how to produce silk-like spider proteins recombinantly. Which I'm sure has a lot of relevance to my life, but anyway, it's kind of cool that you can get random protocols without paying $500 a month. So if you were ever curious about how to make and purify spider silk-like proteins, you're in luck, I guess. I learned this from twitter as well.

On being an o-chem lab TA

It's very funny to me; all of a year ago I was a struggling sophomore messing up the very most basic of things in organic lab. I remember feeling frustrated, feeling like I was bad at lab, feeling like I was one of "those kids" for the prof and the TA.

Now I am a lab TA for organic, and it's all very automatic. I can anticipate the questions before they come; my eye gets drawn to certain very specific mistakes. It's usually things like "no you're sep funnel isn't broken, you just need to uncap it before running it down," or "by the way, your reflux condenser hoses are connected backwards," or "you don't need to heat that--25 degrees is room temperature," or "do more polar things run higher or lower than less polar things on silica gel?" or "is water more or less dense than most organic solvents?" or "you should really put boiling chips in that".

Furthermore, when people ask me for NMR help, it's funny what seems so obvious now. Like common solvent peaks--being able to just identify ethyl acetate or isopropanol or ether. Just knowing what shifts and J-values are characteristic of what.

I guess I can see how some of my professors are the way they are after, you know, 40 years of this.