As part of a research study investigating enzymatic activity in both normal and diseased liver tissue, hepatocytes are isolated from biopsy samples obtained from patients undergoing routine care at a local tertiary referral center. The cells are homogenized and centrifuged to remove membrane components and organelles. Following subsequent rounds of centrifugation, the remaining supernatant contains only cytosol and cytosolic proteins. Activity of which of the following enzymes will most likely be detectable in the supernatant of healthy liver cells?
Enzymes and the biochemical processes they catalyze often require distinct chemical environments for optimal function; small variations in temperature, molecular concentrations, and pH may render them ineffective. Cellular compartmentalization provides a means by which multiple heterogeneous environments can exist within a cell. Organelles such as the nucleus and mitochondria form distinct, membrane-bound regions with a chemical composition different from the cytosol. This allows multiple biochemical processes to occur simultaneously at maximum efficiency.
The cytosol and mitochondria are the predominant sites of metabolism in the cell. Mitochondria are the site of beta-oxidation of fatty acids, the citric acid cycle, and the carboxylation of pyruvate (gluconeogenesis). The cytosol is home to enzymes necessary for glycolysis, fatty acid synthesis, and the pentose phosphate pathway. Transketolase is an enzyme of the pentose phosphate pathway that uses thiamine (vitamin B1) as a cofactor to shuttle 2-carbon fragments between sugar molecules. Other processes such as heme synthesis, the urea cycle, and gluconeogenesis rely on a complex interplay between the mitochondria and cytosol to function optimally; enzymes present in both cellular compartments are required for these metabolic pathways.
(Choice A) 3-Hydroxy-3-methylglutaryl-CoA (HMG CoA) lyase is a mitochondrial enzyme necessary for ketogenesis. It is also responsible for metabolism of the ketogenic amino acid, leucine.
(Choice B) Ornithine transcarbamylase catalyzes the combination of ornithine and carbamoyl phosphate to form citrulline in the urea cycle. This reaction occurs within the mitochondria.
(Choice C) Pyruvate carboxylase catalyzes the initial step in gluconeogenesis by converting pyruvate to oxaloacetate. This enzyme requires biotin as a cofactor, and functions within the mitochondria.
(Choice D) Succinate dehydrogenase is a TCA cycle enzyme that converts succinate to fumarate. It is an inner mitochondrial membrane protein and functions as part of the electron transport chain.
Educational objective:
Cellular compartmentalization allows multiple biochemical processes to occur simultaneously at maximum efficiency. Beta-oxidation of fatty acids, the TCA cycle, and the carboxylation of pyruvate (gluconeogenesis) all occur within the mitochondria. The enzymes responsible for glycolysis, fatty acid synthesis, and the pentose phosphate pathway reside in the cytosol.