Thiopurine S-methyltransferase (TPMT) is an enzyme that is responsible for the metabolism of the drugs azathioprine and 6-mercaptopurine, which are used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Metabolism by TPMT reduces the bioavailability of the drugs. In addition to the thiopurine substrates, TPMT also binds S-adenosyl methionine as a cofactor. TPMT is a highly polymorphic enzyme; almost 30 variants are known to be expressed in humans, with most of the variants possessing lower enzymatic activity than the wild-type protein (TPMTwt). Despite having substitutions remote from the active site, TPMT*2 (an A80P variant) and TPMT*5 (an L49S variant) have enzyme activities of 47% and 14% relative to TPMTwt at 18°C, respectively. Two techniques were used to understand the relationships between enzymatic activity and protein stability. Circular dichroism (CD) spectroscopy measures the differential absorption of left and right-circularly polarized light and is used to examine the secondary structure of proteins. The thermal denaturations of TPMTwt, TPMT*2, and TPMT*5 were followed by CD spectroscopy at 222 nm (Figure 1). Fraction unfolded Figure 1 1.0+ 0.5- 0- To analyze the relative flexibilities of different regions of the TPMT proteins, the proteins were incubated with chymotrypsin, which preferentially cleaves at aromatic and bulky hydrophobic residues, for lengths of time ranging from 0 to 200 min. Ο Α. 20 30 40 50 60 70 80 Temperature (°C) Adapted from P. Wennerstrand et al., Biochemistry ©2012 American Chemical Society. O B. Thermal denaturation of TPMT proteins followed by CD spectroscopy Which physical property does NOT change with the amino acid substitution made in TPMT*5? OD. O A. Molecular weight B. Hydrophobicity OC. Hydrogen bonding capability OD. Net charge Samples from various time points of the proteolysis of TPMTwt were subjected to SDS-PAGE under reducing conditions. Which figure best depicts the expected appearance of the gel? TPMTWt -- TMPT*2 ... TMPT*5 (Note: The arrow indicates the movement of the protein through the gel.) |||| 05 Time (min) 05 50 200 Time (min) || 50 200 0 5 50 200 Time (min) 0 5 50 200 Time (min) A. +15 g/mol. If the combined mass of the TPMT substrate and cofactor was determined before the enzymatically catalyzed reaction and then compared to the combined mass of the product and the cofactor after the reaction, the net change in molecular weight will be: OB. 0 g/mol. O C. -15 g/mol. OD. -16 g/mol. The CD spectroscopy signal that was used to generate the data in Figure 1 arises from the chirality of the: O A. a carbon. B. amide nitrogen. O C. carbonyl carbon. OD. ß carbon.