Tost Mwt Stds, KD 272 132 95 || Non-denaturing Denaturing 11 - 66 - 45 14

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 metabolic pathway.

Following purification of the PYC enzyme by avidin-Sepharose affinity chromatography, the investigators carried out several experiments to characterize the enzyme.

First, they ran samples of the enzyme on denaturing and non-denaturing gels; the results are shown in the figure to the right. In addition, they ran the protein through a calibrated gel filtration column, the results of which indicated that the PYC enzyme had a molecular weight of 540 kiloDaltons. In the figure, the rightmost column are where molecular weight standards of various sizes would occur if they’d been on the gels.

The catalytic properties of the PYC enzyme were assessed following purification. The activity of the enzyme was assayed in the presence of ATP, pyruvate, bicarbonate and Mg²⁺ ions (these latter ions were the control substance). In addition, the dependence of the enzyme on these various metabolites was tested by replacing them with similar compounds. The activity of the enzyme in the presence of these various molecules is shown in the table.

Part A} 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? Would this happen if the amino acid residue were not lysine?