Chapter 4
GTP-binding Proteins and
Signal Transduction
Nucleotides as metabolic regulators
In addition to providing the alphabet of the genetic code, nucleotides play many other roles. The pyrimidine bases act as identifiers for metabolites.
For instance, CTP is a reactant in phospholipid biosynthesis in which the CMP moiety is transferred to phosphatidate in the formation of CDPdiglyceride (see Figure 8.5). Similarly, it reacts with choline phosphate in the formation of CDP-choline. Uridine nucleotides are involved in the assembly of polysaccharides:
UDP-glucose pyrophosphorylase
glucose-1-P UTP →UDP-glucose PPi
Purine nucleotides play their main regulatory roles in association with proteins, not metabolites. ATP acts as a link between metabolic processes and cellular activities. It is present in most cells at high concentration, 5–10 mmol L 1 in the
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Signal Transduction
Estimates of cytosol ATP concentration have been somewhat lower for other (non-muscle) cell types (1–3 mmol L 1). The determination of its concentration depends on a good estimate of cytosol volume.
cytosol of muscle and nerve. It is also subject to rapid turnover. The
human body typically turns over 90% of its entire content of ATP within 90 s; thus, an amount equivalent to 75% of body weight, about 40 kg, is turned over every day. In spite of this, the cellular concentration of ATP generally remains remarkably constant, even in working muscle. In the extracellular environment ATP also acts as a neurotransmitter.
The possibility that GTP might act as a factor necessary for cellular processes first became apparent in the action of phosphoenolpyruvate carboxylase
in gluconeogenesis and for succinyl CoA synthase in the operation of the tricarboxylic acid cycle. It is a necessary component, together with mRNA, activated amino acids (aminoacyl-tRNA), and ribosomes, in the initiation and elongation reactions of protein synthesis. In this sense, the initiation factors and elongation factors should be regarded as the original GTP-binding proteins.
ATP is not quite what it seems
In the early years following the discovery of cAMP, all that was required (beyond a great deal of skill and dedication) to detect the activity of adenylyl cyclase in cell membrane preparations was an activating hormone and the substrate ATP (as its Mg2 salt) (Figure 4.1a). The membranes, containing an appropriate receptor and the catalytic unit, did the rest. At least, that was the general idea.
The economical manufacture of ATP, in a high state of purity and in large quantities, has been a notable achievement of the commercial suppliers. It is now possible to purchase a gram of crystalline ATP, better than 99% pure, for less than the price of a pint of beer, but this has not always been the case. In the 1960s commercially produced ATP was good enough as a substrate, but sometime around 1970 it became erratic, occasionally registering zero activity in the cyclase reaction. A similar impasse had been encountered previously in the investigation of fatty acid biosynthesis. It had been found that an essential impurity was lost as the chemical purity of the ATP was improved. This was CTP (cytidine 5 -triphosphate). In the case of adenylyl cyclase experiments
it was the exclusion of contaminating traces of GTP that caused the loss of activity. Addition of GTP now allowed hormone-induced generation of cAMP to proceed.1 Pertinently, it also had the effect of reducing the affinity of agonist (but not of antagonist) binding at receptors.2–4 It was clear that GTP plays a central role in the cyclase reaction, not as a substrate but as a cofactor. The two established components, in Rodbell’s terminology the discriminator (receptor) and the effector, were now joined by a third, the transducer that binds GTP (Figure 4.1b).
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