2. The kinetics of the ATCase reaction were examined using increasing concentrat

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.

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 ase 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. HCO3 Glutamine ATP ATCase Carbamoyl phosphate Aspartate N-carbamoylaspartate UMP UTP Figure 16.1: Pyrimidine synthetic pathway. CTP

Explanation / Answer

Information from the shape of the curve:

Control: A plot of substrate (aspartate) concentration versus velocity is represented showing a sigmoidal curve. The binding of aspartate to one active site convert the entire enzyme into the R state, which increases the activity of other active sites (cooperativity). So it shows a sigmoidal curve.

With CTP: (T state curve) It allosterically inhibits the ATCase enzyme, making substrate binding difficult, thus stopping it from reaching the R state and thus stabilising Tensed (T) state. Thus, the curve is shifted to the right.

With ATP: It allosterically activate the enzyme thus stabilizing the Relaxed (R) state, shifting the curve to left.

b .ANS:

Kinetic Parameters that change in the presence of CTP: The Km or concentration of substrate for half-saturation, for aspartate is increased for that molecule.

Kinetic Parameters that is unchanged in the presence of CTP: Vmax (maximum rate of reaction) is unchanged.

C. ANS:

Kinetic Parameters that change in the presence of ATP: The Km or concentration of substrate for half-saturation, for aspartate is decreased for that molecule.

Kinetic Parameters that is unchanged in the presence of CTP: Vmax is unchanged.

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