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Tuesday, December 09, 2014

On the meaning of pH optima for enzyme activity

The students in my lab course measured the activity of trypsin at different pH's. They discovered that the enzyme was most active at a pH of about 8.0-8.5 and that activity fell off rapidly at pH values above and below this optimum. This is consistent with results in the published literature (see figure from Sipsos and Merkel, 1970). Here's the exam question ...
What was the pH optimum of trypsin activity? Can you explain this in terms of the normal biological function of the enzyme and the physiological conditions under which it is active? Do you expect there to be a strong correlation between the optimal pH of an enzyme’s activity and the pH of the cell/environment where it is active?

Sipos, T., and Merkel, J. R. (1970) An effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry, 9:2766-2775 [doi: 10.1021/bi00816a003]


Tom Mueller said...

Hi Larry

I am intrigued by your question.

I always taught that the small intestine has a PH of 7 to 9, which is neutral to alkaline… and since the pancreas secretes Trypsin into the duodenum, optimal activity in slightly alkaline conditions would make adaptive good sense.

I typically reinforce this concept by narrating an apocryphal story of UBC engineers baking Phenolphthalein into cookies handed out to students for free…

Phenolphthalein is a potent laxative and the resultant projectile diarrhea was “blood-red” in color, constituting perhaps the nastiest practical joke ever perpetrated on the student body. Of course, to prevent copy-cat crimes, I never identify Phenolphthalein in class.

A quick google-whack indicates my answer may be naïve!

The intraluminal pH is rapidly changed from highly acid in the stomach to about pH 6 in the duodenum. The pH gradually increases in the small intestine from pH 6 to about pH 7.4 in the terminal ileum. The pH drops to 5.7 in the caecum, but again gradually increases, reaching pH 6.7 in the rectum.

In that case… I may need to change my answer!

Perhaps – Trypsin is not “intelligently designed” and typically needs to operate at sub-optimal pH.

That said Enteropeptidase does seem to operate at optimal pH – perhaps indicating that optimization of Enteropeptidase trumps optimization of Trypsin.

In that case – Trypsin’s sub-optimal activity needs to compensated by an extremely low Km and increase in enzyme concentration. That, or something else is happening in the intestinal lumen that stabilizes Trypsin tertiary structure that I am unaware of.

OK... that's as far as I can take it. What is your correct answer?

Tom Mueller said...

@ Joe & @ Larry

Forgive the off-topic post. Just FTR: I have have cited you both (extensively) on

... in response to a champion of ID

As professors in "Biology" you can access these posts by becoming members of the AP community.

I just wanted to declare my debt of gratitude to you both!


Larry Moran said...

It seems to be a popular misconception that the pH of the intestine is alkaline and that accounts for the pH optimum of trypsin. As you (and my students) have discovered, the actual pH of the intestine of not terribly alkaline.

The pH optimum of an enzyme is determined largely by its mechanism of action. In the case of trypsin, we are dealing with the famous "catalytic triad" of aspartate, histidine, and serine. The deprotenation of the serine hydroxyl group is an essential part of the mechanism but serine has a pKa of about 16 under normal circumstances. In the active site this deprotenation is lowered to around 8 or 9 by hydrogen bonding to the adjacent imidazole of histidine.

Because of this mechanism, the optimal pH is in the alkaline range but there's enough activity at pH 6 or 7 to get the job done. The activity of trypsin has evolved to be "good enough" and that's all that's required. There's good evolutionary reasons why an enzyme has to be active at the pH of the normal environment but there's no reason why this has to be the optimal pH for activity.

Tom Mueller said...

Please allow me to repeat myself...

I wish I could sit in on your classes!

Thank you for (yet again) correcting a major misconception on my part.

Athel Cornish-Bowden said...

I hope you make it clear to your students that the paper would fall far short of an acceptable pH dependence study in 2014, and if my D.Phil. supervisor (Jeremy Knowles) had been the referee he’d have been pretty scathing about it in 1970.

What has been measured? OK, something called “activity”, but what’s that? It appears to be a rate, as it is mmol p-Tos-L-ArgOMe min^-1 mg trypsin^-1. But what sort of rate? An initial rate (estimated how?)? An average over some period, maybe whatever the authors thought of as the “linear phase”? The authors don't say.

But, in any case, showing any sort of rate as a function of pH may be useful as a preliminary step towards doing a proper experiment, i.e. estimating some kinetic parameters, ideally rate constants for elementary steps, but at least Michaelis-Menten parameters. Fortunately Jeremy was satisfied in 1967 with Michaelis-Menten parameters (which is what he had me measure), but several years later (around 1972) he was saying that they didn’t mean much either. I think he was probably exagggerating deliberately to get a reaction from his audience, but I was very unhappy when I heard him say that in a lecture.

Another problem with the Sipos/Merkel paper is that they make no attempt to calculate any curves. This was perfectly possible to do in 1970: I had calculated curves in my D.Phil. thesis of 1967.

As to what “alkaline” means, at that time I referred to any buffer above about pH 4 as “alkaline” to my fellow students who were working on boring enzymes, because pepsin loses activity above pH 5. However, don’t quote me on that: I don’t want anyone thinking I really think that a pH 6 solution is an alkali.

Seeing the names on the paper I wondered if one of them has a sister who is now Chancellor of Germany (probably not, but you never know). Also, in the acknowledgements, I read “We are especially grateful to Mrs. R. Dawkins for running the ultracentrifuge experiments”. Not Marian Stamp Dawkins, I assume, as I don’t suppose she has ever run an ultracentrifuge experiment.

Larry Moran said...

I agree with some of your criticisms of the 1970 paper but it wasn't the point. In the lab, the students measured the initial rate as a function of pH and they also calculated Km and kcat. The idea was to show them that you could learn something about mechanism from such data.

This is the first (and last) time that I've taught this lab course. I struggled to find the happy medium between learning and observing some basic concepts and thinking critically about experimental design. I want them to think critically but still see the value in a lab course.

This is harder than it seems because I don't see much value in most lab courses - at least not the way they are currently taught. But I also don't see much value in a lot of research that's being published.

Athel Cornish-Bowden said...

No quarrel with any of that, Larry. I used to hate running laboratory courses when I had to do it. The worst was when I had to run practicals that my olders and betters had designed, and I was expected to use their notes, which they had written without understanding things like buffers. In some cases I rewrote the notes (without telling the authors), but sometimes it was just impossible.

Piotr Gąsiorowski said...

Seeing the names on the paper I wondered if one of them has a sister who is now Chancellor of Germany (probably not, but you never know).

The Chancellor's maiden name was Kasner, not Merkel, so at best the co-author could be a relative of her first husband. She's got a PhD in quantum chemistry. Her current husband, Prof. Joachim Sauer, is also a quantum chemist, and they seem to be bonded together by stable molecular orbitals.

Kasner is a mangled form of the Polish surname Kaźmierczak. Angela Merkel's paternal grandfather was born in Poznań, the town where I live, and she has some family in these parts.

judmarc said...

and they seem to be bonded together by stable molecular orbitals

I am stealing "May you be bonded together by stable molecular orbitals" for my next wedding toast. :-)