Question

Enzyme inhibition case study, Part I

It was about 50 minutes into another long, boring two-hour lecture by Dr. Foster. The tall, thin, gray- haired biochemistry professor was concluding a chapter on enzymes. The final topic was enzyme inhibition. Sarah, an energetic and outgoing junior, was daydreaming about her upcoming weekend at home for a family reunion. She was not really paying attention, but heard the professor going on and on about alcohol dehydrogenase. At last, Dr. Foster turned to the class and asked, “Any questions?” Sarah, hoping to side-track the professor, enthusiastically waved her hand. As soon as she received a nod from Dr. Foster, she began to recount a story that her grandfather had told her.

“My grandfather said that when he was a teenager, alcohols were used as antifreeze in automobiles. A group of kids was hanging out in a friend’s garage. One of them found two bottles of liquid that were marked ‘alcohol.’ He talked everyone else into trying the alcohol. All of the kids became severely ill, but about half of the teens died from poisoning. Someone later found out that one of the bottles contained methanol and the other a mixture of ethanol and methanol. The kids who lived drank the mixture. Grandpa figured since I was a chemistry major, I’d know why half died and half didn’t, but I didn’t know. Do you?”

Although Sarah had planned to side-track the professor, her story played nicely into his lesson plan. After all, the incident was related to the current class topic of enzymes and enzyme inhibition. The clever professor began, “Let me remind all of you that the enzyme, alcohol dehydrogenase, catalyzes the first oxidation step in the case of both ethanol and methanol. Methanol is a very common solvent that is quite toxic. I recall an article from a few years ago that described the largest mass methanol poisoning reported to date. It occurred in Nicaragua. Of the approximately 800 poisonings, about 45 patients died and others suffered blindness (SEMP Biot #412, 2006). I seem to recall that poisoned patients received either ethanol or a drug called Fomepizole as an antidote. I think I have some homework that will help you to understand what happened in your grandfather’s story.”

3. Compare and contrast competitive and noncompetitive inhibition. Show the Michaelis-Menten equation and define all of the variables. Include in your answer the use of Lineweaver-Burk plots in distinguishing between the different types of enzyme inhibition.

Answer 1

Fomepizole is also known as 4-methylpyrazole. This is used to treat methanol and ethylene glycol poisoning. It is a competitive inhibitor of alcohol dehydrogenase, which catalyses the initial step of methanol and ethylene glycol to the toxic products.

Shown in the image is the simplest equation of enzyme kinetics, the michaelis menton equation. V depicts the velocity of the reaction, Vmax is the highest maximum velocity, S is the ssubstrate concentration and Km is the Michealis constant which depicts the substrate concentration at which the reaction velocity is half maximal.

It is difficult to determine Vmax directly from a plot of V against [S] and thus Km cannot be readily determined in this way. Thus Lineweaver burke plot came into picture which is a rearrangement of Michaelis menton equation. Rhis is also known as the double reciprocal plot. inhibitors can be of two types, irreversible and reversible. Reversible inhibitors are of three types, competitive, non-competitive and uncompetitive.

VMax
shown above are the michaelis menton and Lineweaver plots for competitive inhibitor. The inhibitor's structure resembles enzyme's natural substrate and it binds to the the active sitr reversibly. Vmax remains constant and the Km increases. The effect of a competitive inhibitor is reversed by increasing the concentration of substrate. For eg, alcohol dehydrogenase, high dose of ethanol is used to alleviate the effect of methanol because ethanol competitively binds the active site alcohol dehydrogenase.

Non competitive inhibitor. It is also called mixed inhibition. The inhibitor binds a site other than the active site of the enzyme. Inhibitor binds and alters the 3-D configuration and blocks the reaction. Inhibitor can bind to either free enzyme or the ES complex. It is not reversed by increasing the concentration of substrate. The Vmax value decreases, Km stays constant.

Uncompetitive inhibitor, is rarely encountered. Inhibitor binds to distinct site to the active site but as opposed to non competitive, binds only to the ES complex. The apparent Vmax and Km both decreases.