The amino acid sequence of PYC from the methanogen was compared to other pyruvate carboxylase enzymes, and it was discovered that the lysine at position 534 is strictly conserved. Why is this the case? It is a covalent bond between the valerate part of biotin and the lysine side chain. Would this happen if the amino acid residue were not lysine?
Introduction: As one of the reactants in the first reaction of the citric acid cycle, oxaloacetate is an important cellular metabolite. The concentrations of oxaloacetate are tightly regulated.
Different organisms employ different mechanisms to obtain oxaloacetate. In mammals and yeast, oxaloacetate is the product of the pyruvate carboxylase (PYC) reaction. In E. coli, the enzyme phophoenolpyruvate carboxylase (PPC) provides oxaloacetate from phosphoenolpyruvate (PEP) obtained from glucose oxidation in the glycolytic pathway. If glucose is absent and E. coli is using acetate as a carbon source, the glyoxylate pathway serves to generate the needed oxaloacetate. Usually, an organism will employ PPC or PYC, but not both.
Detectable levels of PYC in the methanogenic bacterium Methanobacterium thermautotrophicum had previously not been found, and since PPC had been detected, it was believed that M. thermautotrophicum did not possess PYC. However, in the case described here, microbiologists found that if they added biotin to cultures of the methanogenic bacterium, pyruvate carboxylase activity could be detected. This was a surprising finding, especially since it is known that the methanogen can synthesize its own biotin. However, having identified the presence of the PYC enzyme, the investigators set out to isolate, purify and characterize the enzyme. Purification of the PYC was rather straightforward since the enzyme is soluble and hydrophilic. In addition, the investigators were able to make use of the protein avidin, which binds to biotin with high affinity and specificity.
Objective: The enzyme pyruvate carboxylase is discovered in a bacterium that was thought not to contain it; in this case study, you’ll see how researchers study and characterize the enzyme, and, ultimately, show how it fits into a
Following purification of the PYC enzyme by avidin-Sepharose affinity chromatography, the investigators carried out several experiments to characterize the enzyme.
The amino acid sequence of PYC from the methanogen was compared to other pyruvate carboxylase enzymes, and it was discovered that the lysine at position 534 is strictly conserved. Why is this the case? It is a covalent bond between the valerate part of biotin and the lysine side chain. Would this happen if the amino acid residue were not lysine?
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