ATCase

Aspartate transcarbamoylase (ATCase), the catalyst for the first reaction unique to pyrimidine biosynthesis (Figure 34-7), is feedback-inhibited by cytidine tri-... 

However, we should be very clear what the source of this heating is. If it is heat from nearby components, the ATcase.core may not be that high and our estimate would be overly conservative. 

MM Lactate DH t Alcokol P Gfycerate ATCase catjg, Phosphorylase ... 

Fig. 93. Topology diagrams for the doubly wound and miscellaneous a/p domains illustrated in Figs. 76 through 78. Arrows represent the P strands thin connections lie behind the p sheet and fat ones above it. The darkest upper box surrounds the classic doubly wound sheets successively lighter solid boxes include domains that are progressively less like the classic topology the dotted box encloses the miscellaneous a/P structures. K = kinase P = phospho DH = dehydrogenase ATCase = aspartate transcarbamylase.

Many protein molecules exist in two (or more) different structures of the same molecular mass but with different spatial disposition of constituent atoms. An intensively studied enzyme, aspartate transcarbamoylase (abbreviated ATCase), under some circumstances is in an enzymatically less-active constrained steric arrangement, which is labeled the T form, and under other conditions in the enzymatically very active, structurally more swollen and open conformation, the R form. 

Figure 4.1. Thermodynamic cycles, which illustrate states of enzyme ATCase during calorimetric measurements of enthalpies accompanying the binding of progressively increasing quantities of the substrate analog PALA. At the outset, the ATCase is 100% in the T conformation at the conclusion of the transformation, the ATCase (PALA)6 is 100% in the R conformation and has six bound PALA molecules. When the extent of binding of PALA is between 0 and 6, the extent of conformational conversion is between 0% and 100%. The horizontal broken arrow at the top of the diagram indicates the process that is accompanied by the enthalpy that we want to know. The vertical broken arrow at the right represents a pure...

ENTHALPY, ENTHALPY OF REACTION, AND HEAT CAPACITY TABLE 4.7. Enthalpy Change on Binding PALA to ATCase... 

Aspartate transcarbamoylase (ATCase) from Escherichia coli is the most studied and best known regulatory enzyme. Yates and Pardee (1956) were the first to propose that the activity of ATCase is controlled by end product inhibition. This feedback inhibition was later studied in more detail by Gerhart and Pardee (1961, 1962, 1963). The three-dimensional structure of ATCase was determined by Lip-scombe and his coworkers [Wiley etal. (1971), Wiley and Lipscomb (1968), Warren etal. (1973)]. 

ATCase catalyzes the first step in the biosynthesis of cytidine triphosphate (CTP). The sequence of reactions leading from the reactants, aspartate and carbamoyl phosphate, to CTP is shown in Fig. 8.19. 

Figure 8.20 shows the inhibitory effect of CTP, as well as the activation of ATCase by ATP. Based on measurements by Gerhart and Pardee (1962,1963), the reaction rate is here plotted as a function of the concentration of aspartate. It is clear... 

Figure 8.18. A schematic view of ATCase based on electron micrographs, viewed along the threefold symmetry axis. The outer equilateral triangle has an edge of 145 A. The (almost) inscribed solid triangle with edge 95 A is rotated by 60° relative to the large triangle.

Figure 8.19. Sequence reactions from aspartic acid (AA) and carbamoyl phosphate (CP) to the end product, cytidine triphosphate (CTP). The first reaction is catalyzed by ATCase. The intermediary compounds are N-carbamoyl aspartic acid (N-CAA), L-dihydroorotic acid (L-DHOA), orotic acid (OA), orotidine 5 -phosphate (0-5 -P), uridine 5 -phosphate (U-5 -P), uridine diphosphate (UDP), and uridine triphosphate (UTP).

Binding measurements of the bisubstrate analogue N-(phosphoacetyl)-L-aspartate (PALA) in the presence and absence of ATP were reported by Newell, et al, (1989). Figure 8.21 shows the binding isotherm of ATCase, a plot of 0 = [PALA]t,Qy j/[ATCase] as a function of [PALA]f (at 23 °C and in a buffer solution). 

It is evident that addition of CTP lowers the BI while addition of ATP elevates the BI of the effector-free ATCase. 

Figure 8.21. Binding isotherms of PALA to ATCase in the presence of ATP and CTP. Redrawn with changes from Newell et al. (1989).

The fact that ATP and CTP bind to the same site follows from the observation that adding ATP to the inhibited enzyme by CTP reduces or reverses the inhibition, presumably because ATP competes with CTP for the same site. The fact that CTP binds to an allosteric site (i.e., it is not a competitive inhibitor) follows from the so-called desensitization effect. Addition of mercurials [e.g., p-mercuribenzoate (PMB)] reduces or eliminates the inhibition by CTP. However, it has no effect on the enzymatic activity of ATCase, presumably because the mercurials affect the regulatory subunits but not the catalytic site. As for the mechanism of cooperativity (both positive and negative), it is known that CTP does induce changes in the quaternary structure of the enzyme. 

Structural model for conformational changes in ATCase, based on the X-ray crystallographic investigations of Lipscomb and colleagues. Note that substrates are thought to enter through a channel, and allosteric effectors alter channel accessibility. 

ASYMMETRIC INDUCTION ASYMMETRY PARAMETER ASYMPTOTE ATCase,... 

Regulation of the rate of pyrimidine nucleotide synthesis in bacteria occurs in large part through aspartate transcarbamoylase (ATCase), which catalyzes the first reaction in the sequence and is inhibited by CTP, the end product of the sequence (Fig. 22-36). The bacterial ATCase molecule consists of six catalytic subunits and six regulatory subunits (see Fig. 6-27). The catalytic subunits bind the substrate molecules, and the allosteric subunits bind the allosteric inhibitor, CTP. The entire ATCase molecule, as well as its subunits, exists in two conformations, active and inactive. When CTP is... 

It is not easy to mimic the shuffling of domains in vitro by manipulation of genes. For example, each catalytic polypeptide chain of the multimeric E. coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helixes. The E. coli enzyme ornithine transcarbamoylase (OTCase) is 32% identical in sequence and thus of presumably similar structure (see section D8). None of the chimeras in which a domain from one enzyme was attached to the corresponding partner in the other is active. The specific intrachain and interchain side-chain interactions also have to evolve for the Correcting packing.32... 

Although the Michaelis-Menten model provides a very good model of the experimental data for many enzymes, a few enzymes do not conform to Michaelis-Menten kinetics. These enzymes, such as aspartate transcarbamoylase (ATCase), are called allosteric enzymes (see Topic C5). 

Aspartate transcarbamoylase (aspartate carbamoyltransferase ATCase), a key enzyme in pyrimidine biosynthesis (see Topic FI), provides a good example of allosteric regulation. ATCase catalyzes the formation of N-carbamoylaspar-tate from aspartate and carbamoyl phosphate, and is the committed step in pyrimidine biosynthesis (Fig. 2). The binding of the two substrates aspartate and carbamoyl phosphate is cooperative, as shown by the sigmoidal curve of V0 against substrate concentration (Fig. 3). 

Fig. 2. Formation of N-carbamoylaspartate by aspartate transcarbamoylase (ATCase) is the committed step in pyrimidine biosynthesis and a key control point.

ATCase aspartate transcarbamoylase FAD flavin adenine dinucleotide... 

ATCase and ornithine carbamoyltransferase (OTCase) catalyze analogous reactions. ATCase transfers the carbamoyl moiety from carbamoyl phosphate to aspartate, and OTCase transfers the carbamoyl moiety from carbamoyl phosphate to ornithine. They both share a common N-terminal functional domain, which binds carbamoyl phosphate. The C-terminal domains of these enzymes are structurally similar but have... 

In this experiment we will examine some of the properties of the aspartate transcarbamylase of Escherichia coli, which is typical of many enzymes subject to feedback inhibition and which has been studied extensively. Aspartate transcarbamylase (ATCase) catalyzes the first reaction unique to the biosynthesis of pyrimidine nucleotides. ATCase is subject to specific inhibition by quite low concentrations of one of its end products, cytidine 5 -triphosphate (CTP). This relationship and two other regulatory interactions important to the control of pyrimidine biosynthesis are summarized in Figure 9-1. 

The study of the kinetic properties of the ATCase reaction and of its inhibition by CTP, some... 

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