In the Walker/Karmen method for ALT determination, which substrate is used by the enzyme?

• A. Pyruvate
• B. Oxaloacetate
• C. Alpha-ketoglutarate
• D. Malate

Answer: (A)

Transaminases need an amino-donor substrate to transfer an amino group to an α-keto acid. In the ALT (alanine aminotransferase) assay used by the Walker/Karmen method, the enzyme uses L-alanine as the amino donor and α-ketoglutarate as the acceptor to form pyruvate and glutamate. The assay then measures the amount of pyruvate produced by coupling it to lactate dehydrogenase with NADH; the decrease in NADH reflects ALT activity. So the substrate is L-alanine, not pyruvate. Pyruvate is the product generated in this setup. The other compounds mentioned are involved with other transaminases or roles in different parts of metabolic reactions, not the substrate in this ALT assay.