Case 16 Allosteric Regulation of ATCase

Transcription

1 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. rerequisites roperties 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 UT and CT. The enzyme catalyzes the condensation of carbamoyl phosphate and aspartate to form carbamoyl aspartate. The reaction pathway is shown in Figure 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 an early enzyme in a multi-step pathway, the ATCase reaction is a logical one to regulate the synthesis of pyrimidine nucleotides. Both purine nucleotides and pyrimidine nucleotides are needed in roughly equal amounts as substrates for DA 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. C Glutamine + AT ATCase Carbamoyl phosphate + Aspartate -carbamoylaspartate UM UT Figure 16.1: yrimidine synthetic pathway. CT 1

2 CASE 16 Allosteric Regulation of ATCase Table 16.1: ames and Abbreviations of ucleic Acid Bases, ucleotides and ucleosides. (Based on Voet and Voet, 1995.) Base formula Base ucleoside ucleotide ucleotide = = ribose ucleotide - = ribose phosphate = ribose triphosphate = deoxyribose triphosphate 2 Adenine Adenosine AM AT dat 2 Guanine Guanosine GM GT dgt 2 Cytosine Cytidine CM CT dct Uracil Uridine UM UT

3 Questions 1. Gerhart and ardee measured ATCase activity in the presence of a variety of purine and pyrimidine derivatives. Their results are presented in Table 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 Table 16.2: Effect of nitrogen bases, nucleosides and nucleotides on ATCase activity. *Indicates stimulation. (Based on Gerhart and ardee, 1962.) Compound Inhibition, % (Conc = 2 mm) Cytosine 0 Cytidine 24 CM 38 dcm 48 CT 86 dct 88 UT 8 GT 35 dgt 31 AT -180* dat -162*

4 % Activity Velocity, activity units/mg protein CASE 16 Allosteric Regulation of ATCase 2. The kinetics of the ATCase reaction were examined using increasing concentrations of aspartate, in the presence and absence of CT and AT as shown in Figure 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 CT? What parameter(s) do not change? What is the significance of these observations? c. Answer question 2b for AT. Figure 16.2: Kinetics of of ATCase in activity the presence in the presence of AT and of CT CT and (based AT (based on Gerhart on Gerhart and ardee, and ardee, 1962). 1962). 3. The investigators 5 examined the behavior of the ATCase enzyme in 4 the presence of CT, and in the presence of both CT and AT. The 3 concentration of CT is 0.1 mm and the concentration of AT is 2 mm. The 2 results are shown in Figure What 1 is the significance of these observations? Aspartate concentration, mm Control With CT With AT control + CT +CT +AT Figure 16.3: ercent ATCase activity in the presence of CT, and in the presence of both CT and AT (based on Gerhart and ardee, 1962).

5 4. Back in 1962, Gerhart and ardee developed a model for the regulation of the activity of the ATCase enzyme by CT and AT, using the pathway given in Figure 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 CT. They noted that CT did indeed inhibit ATCase, but that the inhibition was always incomplete, even at high concentrations of CT. They hypothesized that perhaps CT 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 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. ow 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. ucleotide Effector Aspartate Concentration 2.5 mm 5.0 mm AT CT GT UT AT/CT AT/GT AT/UT CT/GT CT/UT GT/UT

6 CASE 16 Allosteric Regulation of ATCase References Gerhart, J. C., and ardee, A. B. (1962) J. Biol. Chem. 237, pp Voet, D., and Voet, J. (1995) Biochemistry, John Wiley & Sons, ew York, p Wild, J. R., Loughrey-Chen, S. J., and Corder, T. S. (1989) roc. atl. Acad. Sci. 86, pp