Re: how does low pH affect copper ion inhibition?

Hi Natalie,

It turns out there might be several explanations.

I did a little poking around and found the following abstract at http://entc.allenpress.com/entconline/?request=get-document&doi=10.1897%2F1551-5028(2002) 021%3C1243:IODEAB%3E2.0.CO%3B2

INHIBITION OF DIGESTIVE ENZYME ACTIVITIES BY COPPER IN THE GUTS OF VARIOUS MARINE BENTHIC INVERTEBRATES
Issn: 1552-8618
Journal: Environmental Toxicology and Chemistry
Volume: 21 Issue: 6 Pages: 1243-1248
Authors: Chen, Zhen, Mayer, Lawrence M., Weston, Donald P., Bock, Michael J., Jumars, Peter A.
Article ID:10.1897/1551-5028(2002)021<1243:IODEAB>2.0.CO;2

In the abstract is the following:
"Copper was less effective at inhibiting enzymes at lower pH, suggesting that protons can compete with Cu ion for binding to enzymatically active sites or that enzyme conformation is less vulnerable to Cu inhibition at lower pH."

Both these reasons sound plausible. At low pH, the copper ions would be more soluble in water due to less formation of insoluble copper hydroxides, so it's not a solubility issue, but rather, it's related to how the copper ions interact with the enzyme (or substrate) at different pH.

I read that pepsin has two aspartate residues in its active site that are essential to catalysis. These aspartate residues have different pKa values. At very low pH, both will be protonated. As pH is raised, one will ionize first, then as pH is raised further, the other will ionize too. When they are ionized, they have negative charges that will be attractive to Cu++ ions. So the pH effect could be as simple as this. I think this explanation has more power than the one above, which is vaguer.

I found another interesting paper from 1969 that you can read: http://www.jbc.org/cgi/reprint/244/1/154

This paper discusses inhibition of pepsin by diazo compounds. The inhibition is accelerated by certain metal ions, including Cu++. This paper also finds that inhibition by the diazo compounds is stronger at higher pH, but the reason appears to lie in the reaction of copper ion with the diazo inhibitor, not in the reaction or binding of Cu++ with pepsin.

Regarding alcalase (also spelled “alkalase,” short for “alkaline protease”), this is a subtilisin serine endopeptidase. Its active residues are a histidine, an aspartate, and a serine. Given the aspartate, I would say that the same explanations for pepsin could apply to alcalase as well.

Clearly, this is a topic that has been looked at from many angles in the past. I hope my answer is of help.

-Alex Tobias