Case 16 Allosteric Regulation of ATCase Focus concept An enzyme involved in nucleotide synthesis is subject to regulation by a variety of combinations of nucleotides. Prerequisites Properties of allosteric enzymes. Basic mechanisms involving regulation of metabolic pathways. Background Aspartate transcarbamoylase (ATCase) catalyzes an early step in the synthesis of the pyrimidine nucleotides UTP and CTP. The enzyme catalyzes the condensation of carbamoyl phosphate and aspartate to form carbamoyl aspartate. The reaction pathway is shown in Figure 16.1. The enzyme has been fairly well characterized. It is known to consist of six regulatory subunits and six catalytic subunits. In this case, we examine the properties of ATCase isolated from E. coli to illustrate some of the important regulatory properties of multi-subunit enzymes. As the first enzyme in a multi-step pathway, the ATCase reaction is a logical one to regulate the synthesis of pyrimidine nucleotides. Both purine (ATP and GTP) nucleotides and pyrimidine nucleotides are needed in roughly equal amounts as substrates for DNA synthesis in rapidly dividing cells. The regulation of the ATCase enzyme ensures a proper balance of purine and pyrimidine pools in E. coli. The goal in this case was to identify the cellular metabolites that serve as activators and inhibitors of ATCase. Figure 16.1: Pyrimidine synthetic pathway. 1
Table 16.1: Names and Abbreviations of Nucleic Acid Bases, Nucleotides and Nucleosides. (Based on Voet and Voet, 1995.) Base formula Base Nucleoside Nucleotide Nucleotide Nucleotide X = H X = ribose X = ribose phosphate X = ribose triphosphate X = deoxyribose triphosphate Adenine Adenosine AMP ATP datp Guanine Guanosine GMP GTP dgtp Cytosine Cytidine CMP CTP dctp Uracil Uridine UMP UTP 2
Questions 1. Gerhart and Pardee measured ATCase activity in the presence of a variety of purine and pyrimidine derivatives. Their results are presented in Table 16.2. What compound(s) were the most effective inhibitors? activators? Explain the significance of the metabolites that served as inhibitors or activators in the context of the biosynthetic pathway presented in Figure 16.1. Table 16.2: Effect of nitrogen bases, nucleosides and nucleotides on ATCase activity. *Indicates stimulation. (Based on Gerhart and Pardee, 1962.) Compound Inhibition, % (Conc = 2 mm) Cytosine 0 Cytidine 24 CMP 38 dcmp 48 CTP 86 dctp 88 UTP 8 GTP 35 dgtp 31 ATP -180* datp -162* 3
Figure 16.2: Kinetics of ATCase in the presence of ATP and CTP (based on Gerhart and Pardee, 1962). 2. The kinetics of the ATCase reaction were examined using increasing concentrations of aspartate, in the presence and absence of CTP and ATP as shown in Figure 16.2. a. What information can you obtain by looking at the shapes of the curves in this figure? b. What kinetic parameter(s) change in the presence of CTP? What parameter(s) do not change? What is the significance of these observations? c. Answer question 2b for ATP. 3. The investigators examined the behavior of the ATCase enzyme in the presence of CTP, and in the presence of both CTP and ATP. The concentration of CTP is 0.1 mm and the concentration of ATP is 2 mm. The results are shown in Figure 16.3. What is the significance of these observations? Figure 16.3: Percent ATCase activity in the presence of CTP, and in the presence of both CTP and ATP (based on Gerhart and Pardee, 1962). 4
4. Back in 1962, Gerhart and Pardee developed a model for the regulation of the activity of the ATCase enzyme by CTP and ATP, using the pathway given in Figure 16.1. Describe that model, using information presented here as well as what you have learned about allosteric enzymes. Be sure to include a sentence explaining the physiological significance of your model. 5. Many years later, in 1989, Wild, et al. revisited the idea of allosteric control of ATCase by CTP. They noted that CTP did indeed inhibit ATCase, but that the inhibition was always incomplete, even at high concentrations of CTP. They hypothesized that perhaps CTP did not act alone, but in combination with some other nucleotide. They tested the activity of ATCase in the presence of several nucleotide combinations. The results are shown in Table 16.3. a. What combination gives the most effective inhibition? b. What is the physiological significance of this combination? c. Revise Figure 16.1 to include this new information. d. Redraw Figure 16.2 to include this new information. How does the K M of the nucleotide combination compare with the values for the nucleotides alone? Table 16.3: Relative specific activities for combinations of nucleotide effectors. A value greater than one indicates stimulation, a value less than one indicates inhibition. Nucleotide Effector Aspartate Concentration 2.5 mm 5.0 mm ATP 1.86 1.35 CTP 0.31 0.43 GTP 0.57 0.71 UTP 0.95 0.95 ATP/CTP 0.70 0.85 ATP/GTP 1.98 1.58 ATP/UTP 1.96 1.52 CTP/GTP 0.41 0.58 CTP/UTP 0.05 0.06 GTP/UTP 0.66 0.84 5
References Gerhart, J. C., and Pardee, A. B. (1962) J. Biol. Chem. 237, pp. 891-896. Voet, D., and Voet, J. (1995) Biochemistry, John Wiley & Sons, New York, p. 191-194. Wild, J. R., Loughrey-Chen, S. J., and Corder, T. S. (1989) Proc. Natl. Acad. Sci. 86, pp. 46-50. 6