Fermentation is an important mechanism for generating ATP in anaerobic conditions, when the cell cannot undergo cellular respiration. In yeast, fermentation is a key step in the beer making process. During fermentation, yeast converts sugars present in the pre-beer mixture into alcohol. This occurs in the absence of oxygen, and produces carbon dioxide as a by-product.
Glycolysis is the first step in this process, where the sugar molecules are broken down into pyruvate. Pyruvate then gets decarboxylated by the enzyme pyruvate decarboxylase. Pyruvate decarboxylase requires two co-factors during catalysis, magnesium and thiamine pyrophosphate (TPP), a vitamin B1 derivative. TPP has a thiazole ring, which is capable of forming a carbanion that nucleophilically attacks the substrate during pyruvate decarboxylase catalysis. The products of the decarboxylation reaction are acetaldehyde and carbon dioxide, which is released as a by-product of the reaction. Next, alcohol dehydrogenase converts acetaldehyde into ethanol. This step requires the co-factor nicotinamide adenine dinucleotide (NADH), which gets oxidized to NAD+ in the process.
Humans undergo fermentation in anaerobic conditions, for example, when muscles run out of oxygen during exercise. However, unlike in yeast cells, pyruvate gets catalyzed into lactic acid by the enzyme lactate dehydrogenase. This reaction also oxidizes NADH to NAD+. Lactic acid buildup is responsible for muscle fatigue, and, as muscles do not have a way to metabolize lactic acid, lactic acid is mainly removed by the bloodstream and subsequently broken down in the liver.
Figure 1. UV absorption spectra of NAD+ and NADH
Attribution: "NADNADH" by Cronholm144, Wikimedia Commons.
Figure 2. Reaction coordinate diagram of the lactate dehydrogenase reaction.
If alcohol dehydrogenase, acetaldehyde, and NADH are mixed together in a reaction, which of the following measurements would be the best way to determine the activity of the enzyme?
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