Regulatory enzymes competitive binding

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. 

Three important classes of inhibitors are shown in Table 1-8-3. Competitive inhibitors resemble the substrate and compete for binding to the active site of the enzyme. Noncompetitive inhibitors do not bind at the active site. They bind to regulatory sites on the enzyme. Irreversible inhibitors inactivate the enzyme similar to removing enzyme from the assay. 

The mechanisms by which antitumor-promoters suppress the tumor promotion are not known, but may be due to the following effects (i) inhibition of polyamine metabolism (ii) inhibition of arachidonic acid metabolism (iii) protease inhibition (iv) induction of differentiation (v) inhibition of oncogene expression (vi) inhibition of PKC and (vii) inhibition of oxidative DNA damage [3,6,91]. The polyamine content of cells is correlated to their proliferative, and often, their neoplastic capabilities. A key enzyme in the polyamine biosynthetic pathway, ornithine decarboxylase (ODC), catalyzes the convertion of ornithine to putrescine. Phorbol ester promoters such as TPA cause increased ODC activity and accumulation of polyamines in affected tissues. Diacylglycerol activated PKC, and the potent tumor promoter, TPA, binds to, and activates PKC, in competition with diacylglycerol. PKC stimulation results in phosphorylation of regulatory proteins that affect cell proliferation. Some chemopreventive agents have inhibitory activity towards PKC. Refer to recent review articles for further discussion [3,6,91]. 

Protein kinase C (PKC) actually constitutes a family of several isozymes that are activated by diacylglycerol (DG). The potent tumor promoter, TPA, binds to, and activates PKC, in competition with DG. PKC stimulation results in phosphorylation of regulatory proteins that affect cell proliferation. There is evidence that carcinogenesis may be suppressed by inhibiting this enzyme. Phorbol tumor promoters such as TPA can replace DG in activating PKC. Chemicals that inhibit PKC also inhibit TPA-induced tumor promotion in mouse skin [23]. 

Aspartate carbamoyltransferase catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate in the first committed step of pyrimidine biosynthesis (Chap. 15). The enzyme from the bacterium E. coli (Mr = 310,000) consists of 12 subunits, six regulatory and six catalytic. CTP is a negative effector i.e., it inhibits the enzyme, and does so through binding to the regulatory subunits. ATP is a positive effector that acts through the regulatory subunits, while succinate inhibits the reaction by direct competition with aspartate at the active site (see Chap. 9 for more on effectors). 

The enzyme contains two catalytic sites, two regulatory sites and two specificity sites. The catalytic site binds the substrates, thioredoxin (reduced by NADPH + H+) and the nucleoside diphosphates. The allosteric regulatory site binds ATP as an activator in competition with dATP as an inhibitor. The specificity site binds dGTP, dTTP and dATP but not dCTP and modulates ribonucleotide reductase activity selectively for the four NDP substrates to balance the four dNTP pools. 

The answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

Garthwaite et al. [29] developed a competitive ELISA based on rabbit antibodies and a breve-toxin/peroxidase conjugate, hi this assay, antibodies were coated on the plate, and free toxin in the sample competed with peroxidase-labeled toxin for antibody binding. Upon addition of enzyme substrate, a concentration-dependent reduction in color development was observed. Initial assay performance was modest, but careful optimization of assay parameters later resulted in reduced assay time (1-2 h) and a working range of approximately 0.5-17 ng/mL. This assay has been used extensively in New Zealand to evaluate toxin production in G. breve cell cultures as well as regulatory screening of shellfish. 

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