Fructose bisphosphate aldolase in animal muscle is a class I aldolase, which forms a Schiff base intermediate between substrate (for example, fructose- 1,6 -bisphosphate or dihydroxyacetone phosphate and a lysine at the active site (see Figure 18.12 ). The chemical evidence for this intermediate comes from studies with aldolase and the reducing agent sodium borohydride, \(\mathrm{NaBH}_{4}\). Incubation of the enzyme with dihydroxyacetone phosphate and \(\mathrm{NaBH}_{4}\) inactivates the enzyme. Interestingly, no inactivation is observed if \(\mathrm{NaBH}_{4}\) is added to the enzyme in the absence of substrate. Write a mechanism that explains these observations and provides evidence for the formation of a Schiff base intermediate in the aldolase reaction.

Step by step solution

01

Formation of Schiff Base with Substrate

The class I aldolase enzyme reacts with its substrate, in this case either fructose-1,6-bisphosphate or dihydroxyacetone phosphate, forming a Schiff base at the active site. This involves a lysine at the active site of the enzyme that forms an imine intermediate with the carbonyl group of the substrate.

02

Understanding the Role of the Reducing Agent

Sodium borohydride (\(\mathrm{NaBH}_{4}\)) is a strong reducing agent that can reduce imines to amines. In the absence of substrate, there is no imine to reduce and so no effect on the enzyme’s activity is observed when \(\mathrm{NaBH}_{4}\) is added to aldolase alone.

03

Reduction of the Schiff Base and Enzyme Inactivation

When aldolase is incubated with substrate and \(\mathrm{NaBH}_{4}\), the Schiff base (imine) intermediate is reduced by \(\mathrm{NaBH}_{4}\) to form an amine. This inactivates the enzyme since it no longer has the functional lysine necessary for catalysis.

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