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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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October 29, 2007

What We Don't Know About Enzymes

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Posted by Derek

There was an intriguing paper published earlier this month from Manfred Reetz and co-workers at the Max Planck Institute. It's not only an interesting finding, but a good example of making lemonade from lemons.

They were looking at an enzyme called tHisF, a thermostable beast from a marine microorganism that's normally involved in histamine synthesis. It has an acid/base catalytic site, so Reetz's group, which has long been involved in pushing enzymes to do more than they usually do, was interested in seeing if this one would act as an esterase/hydrolase.

And so it did - not as efficiently as a real esterase, but not too shabby when given some generic nitrophenyl esters to chew on. There was some structure-activity trend at work: the larger the alkyl portion of the ester, the less the enzyme liked it. Given a racemic starting material, it did a good job of resolution, spitting out the R alcohol well over the S isomer. All just the sort of thing you'd expect from a normal enzyme.

Next, they used the crystal structure of the protein and previous work on the active site to see which amino acids were important for the esterase activity. And here's where the wheels came off. They did a series of amputations to all the active side chains, hacking aspartic acids and cysteines down to plain old alanine. And none of it did a thing. To what was no doubt a room full of shocked expressions, the enzyme kept rolling along exactly as before, even with what were supposed to be its key parts missing.

Further experiments confirmed that the active site actually seems to have nothing at all to do with the hydrolase activity. So what's doing it? They're not sure, but there must be some other non-obvious site that's capable of acting like a completely different enzyme. I'm sure that they're actively searching for it now, probably by doing a list of likely point mutations until they finally hit something that stops the thing.

So how often does this sort of thing happen? Are there other enzymes with "active sites" that no one's ever recognized? If so, do these have any physiological relevance? No one knows yet, but a whole new area of enzymology may have been opened up. I look forward to seeing more publications on this, and I'll enjoy them all the more knowing that they came from a series of frustrating, head-scratching "failed" experiments. Instead of pouring things into the waste can, Reetz and his co-workers stayed the course, and my hat's off to them.

Comments (10) + TrackBacks (0) | Category: Biological News


1. Peter Ellis on October 30, 2007 2:50 AM writes...

I do hope they ran a parallel control series without any enzyme in, just to make sure it's not some other random component of their reaction mix that's doing the business.

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2. John Spevacek on October 30, 2007 8:08 AM writes...

"Promiscuity" in an article title? They'll never let that through the firewall here at work.

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3. qetzal on October 30, 2007 8:55 AM writes...

I can just imagine the lab meetings:

"This has to be contamination. Go back and do everything all over again. Make sure you use all new reagents, fresh buffers, triple clean all the glassware, pour all new columns for purifying the enzyme, resequence the mutant clones...."

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4. Derek Lowe on October 30, 2007 8:57 AM writes...

The problem with using a term like "promiscuity" to bring in random web traffic is that the people who would be inclined to Google it generally can't spell it well enough to find your article. . .

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5. Wavefunction on October 30, 2007 4:43 PM writes...

But of course promiscuous binders is now common phraseology, so firewalls will have to be tweaked to accomodate it.

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6. Milo on October 30, 2007 7:33 PM writes...

The exact same thing happened to my postdoc lab (with another enzyme) while I was there. We were all puzzled and confused. But that is really the essence of science, right?

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7. leftscienceawhileago on October 30, 2007 10:23 PM writes...

I remember originally being wowed by Hellinga's earlier work of doing things like engineering triosephosphate isomerase activity into unrelated proteins thinking "well the rest of us may as well go home!".

AFAIK no structures exist of those proteins (despite the work being predicated on crystal structures)...I always thought that they would see that the structural predictions they were making had very little to do with the activity they were is good to see such a well documented case.

I wonder if we start seeing many small labs pull out some of their old mutants and start publishing similar observations; we may get a new picture on just what "promiscuous" means in terms of enzyme activity.

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8. srp on October 31, 2007 8:11 PM writes...

So is this like a can opener with a previously unknown bottle opener on the other end? Where people used to think the can opener was also opening the bottles?

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9. Bryan on November 1, 2007 1:51 PM writes...

I agree with Ellis in post #1. The MS and gels don't look kosher to me. Ghost bands all over the coomassie stained gels.

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10. Jonadab the Unsightly One on November 10, 2007 10:10 AM writes...

I would not be at all surprised if it turns out that *most* enzymes have multiple active sites and multiple roles. Biological systems are nothing if not complicated.

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