(ASKING FOR QUESTION NUMBER 2.) Background Aspartate transcarbamoylase (ATCase)
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Question
(ASKING FOR QUESTION NUMBER 2.)
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 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 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.
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.
QUESTION 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.
figure 2 and 3
HCO3 Glutamine ATP ATCase Carbamoyl phosphate Aspartate N-carbamoylasparitate UMP UTP Figure 16.1: Pyrimidine synthetic pathway. CTPExplanation / Answer
QUESTION 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?
Answer: ATP acts as a positive effector of ATCase, and CTP acts as an inhibitor. Aspartate (substrate) homotropically regulates ATCase activity, providing sigmoidal kinetics. CTP, an end product, heterotropically inhibits, but ATP, a precursor, heterotropically activates ATCase. In the ATCase reaction, inhibition by CTP and activation by ATP are both heterotropic effects. When CTP is in short supply in an organism, the ATCase reaction is not inhibited, and the binding of ATP increases the activity of the enzyme still more.
b. What kinetic parameter(s) change in the presence of CTP? What parameter(s) do not change? What is the significance of these observations?
CTP acts as an inhibitor of ATCase, CTP increases the initial phase of the sigmoidal curve. In the presence of CTP, the enzyme becomes less responsive to the cooperative effects facilitated by substrate binding; more substrate is required to attain a given reaction rate. At high substrate concentrations, the same maximal rate, Vmax, is observed in the presence and absence of inhibitor.
c. Answer question 2b for ATP.
ATP acts as a positive effector of ATCase, the effect of ATP is to increase the reaction rate at a given aspartate concentration. At high concentrations of ATP, the kinetic profile shows a less pronounced sigmoidal behavior. In the presence of ATP, the rate of the enzymatic reaction is increased at lower levels of aspartate, and the shape of the rate curve becomes less sigmoidal and more hyperbolic. It shows that here is less cooperativity in the reaction. When CTP is in short supply in an organism, the ATCase reaction is not inhibited, and the binding of ATP increases the activity of the enzyme still more.
ATCase is an example of an enzyme system where it is not dealing with a Michaelis–Menten type of enzyme and the term KM is not applicable.
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