Lactase Activity Under Two Buffers
A student homogenizes 2.0 g of calf intestinal mucosa in 20 mL of cold buffer and divides the filtrate into two tubes. Tube A is brought to pH 6.0; Tube B to pH 8.0. Both are kept at 37 °C. At t = 0, 5.0 mL of 0.10 M lactose is added to each tube. At 10-minute intervals, 0.5 mL aliquots are removed and assayed for glucose. Tube A glucose rises steadily and plateaus by 40 minutes. Tube B glucose rises for 10 minutes, then stops changing, even though lactose remains detectable in the tube. When the student adds more lactose to Tube B, no further glucose appears. When the student adds a small volume of the pH 6.0 buffer to Tube B, glucose production resumes briefly and then stops again.
1. Propose the most likely explanation for the difference between the two tubes, and explain why adding lactose alone did not restore activity in Tube B.
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The mucosa homogenate contains lactase, whose activity depends on protonation state of ionizable residues in its active site. At pH 8.0, the enzyme's active-site residues are deprotonated, so substrate binding and/or catalysis is impaired - the enzyme is largely inactive, not destroyed. Adding more substrate cannot rescue catalysis if the active site cannot stabilize the transition state; substrate concentration is not the limiting factor. This is why more lactose does nothing: the enzyme, not the substrate, is the constraint.
2. Explain the transient resumption of activity after adding pH 6.0 buffer, and predict what would happen if the student instead boiled Tube B before adding buffer.
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Adding acidic buffer lowers the local pH, reprotonating the active-site residues and restoring a catalytically competent conformation, so glucose production resumes. The effect is brief because the added buffer is diluted/diffuses and the bulk pH drifts back toward 8.0, inactivating the enzyme again. If Tube B were boiled first, the heat would denature the enzyme (disrupting noncovalent tertiary structure), and lowering the pH afterward would not restore activity - denaturation is not reversible by simple protonation. So no glucose would appear.
3. The student claims the plateau in Tube A proves the enzyme was denatured. Evaluate this claim.
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The claim is not supported. A plateau in product can arise from substrate depletion (lactose fully hydrolyzed) or product inhibition, not only from enzyme inactivation. To distinguish, the student should assay residual lactose at the plateau, or add fresh lactose to Tube A: if glucose rises again, the enzyme was still active and the plateau reflected substrate limitation. Denaturation would be supported only if activity failed to recover after adding substrate and after confirming enzyme was still present.