Enzymes with Short Half-Lives

Enzymes present in the liver cytosol with short half-lives include ornithine decarboxylase, thymidine kinase, tyrosine aminotransferase, tryptophan oxygenase, hydroxymethylglutaryl-CoA reductase, serine dehydratase, and phosphoenolpyruvate carboxykinase. All of these enzymes have degradation rate constants greater than 0.1/h—more than 10 times more rapid than the average ka for liver cytosol proteins (Schimke, 1970). Perhaps a scrutiny of the group can provide information on the enzyme properties as well as the nature of reactions catalyzed by enzymes with rapid turnover rates. 

Without exception, the enzymes are adaptable, often showing 20-fold induction or more to an appropriate stimulus. Indeed, the degree of adaptation shown by these enzymes far exceeds that shown by any enzymes of long half-life listed in Table 1. 

Tyrosine aminotransferase and phosphoenolpyruvate carboxyki-nase are the only enzymes of the group which catalyze readily reversible reactions, and, even with these enzymes, reversibility would not occur to any extent in vivo. Accordingly, the enzymes may be consid- 

As is especially appropriate, for regulatory enzymes, all either catalyze the initial reaction in a metabolic pathway or the first reaction committed to a particular pathway (e.g., phosphoenolpyruvate carboxykinase and hydroxymethylglutaryl-CoA reductase). The enzymes are at points of regulatory control where an increase in activity should result in an increase in substrate throughput. In the basal state, each enzyme is present at a low activity. 

Ornithine decarboxylase and thymidine kinase are associated with cell division and DNA synthesis, i.e., growth. Although the exact biological function of ornithine decarboxylase is unknown, we can assume 

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Although insulin decreases protein breakdown in cultured cells, it has no effect on the degradation rate constants of two enzymes with short half-lives, phosphoenolpyruvate carboxykinase (Gunn et al., 1976) and tyrosine aminotransferase (Reel et al., 1970). This negative result is in keeping with the concept that insulin acts by reducing auto-phagy, a process that seems trivial in the breakdown of proteins with short half-lives (Knowles and Ballard, 1976). 

If the cholesterol 7a-hydroxylating system is of regulatory importance, a short half-life of the enzyme can be expected. Already in 1968 Einarsson and Johansson obtained evidence that this is the case [87]. It was calculated that the half-life for the breakdown of the 7a-hydroxylase was only 2-3 h. Other enzyme activities involved in the biosynthesis of bile acids from cholesterol, such as the 12a-hydroxylase, had a considerably longer half-life time. It seems likely that a cytochrome P-450 species with short half-life time is the component of the cholesterol 7a-hydroxylase. This... 

ACh is metabolised extraneuronally by the enzyme acetylcholinesterase, to reform precursor choline and acetate. Blocking its activity with various anticholinesterases has been widely investigated and some improvement in memory noted. Such studies have invariably used reversible inhibition because of the toxicity associated with long-term irreversible inhibition of the enzyme. Physostigmine was the pilot drug. It is known to improve memory in animals and some small effects have been seen in humans (reduces number of mistakes in word-recall tests rather than number of words recalled), but it really needs to be given intravenously and has a very short half-life (30 min). 

The first evidence that supplementation of exogenous ADA may be helpful in SCID patients came from the 1975 report of Potmar et al. [8], demonstrating that addition of bovine-intestinal ADA or human-erythrocyte ADA to cultures of lymphocytes of a SCID patient restored their ability to proliferate when stimulated with mitogens. The ability to respond to mitogens is an indicator of immune function restoration. Therapeutic use of enzyme extracted from calf tissue revealed that this form of ADA has a short half-life. 

Adenosine deaminase (ADA) was the first therapeutic enzyme coupled to PEG with the aim of reducing clearance and thereby overcoming the short half-life of ADA. Patients deficient in ADA are unable to regulate purine metabolism. As a result purine metabolites (e.g., adenosine monophosphate) accumulate to cytotoxic levels in B-lymphocytes and lead to severe B-cell depletion that presents clinically as severe combined immunodeficiency syndrome (SCIDS). While intramuscular injection of unmodified ADA provides some relief, antibodies develop rapidly against the protein and prevent it from being useful as replacement therapy. Even in the absence of antibodies, unmodified ADA s plasma half-life is only a few minutes. 

The end products of gene expression are proteins, mainly enzymes, and it is essential that their levels be strictly controlled. There are many potential sites of control in both bacteria and eukaryotes. DNA or gene amplification (Chap. 16) in eukaryotes is one way of responding to the demand for more of the protein product if there arc more copies of the gene, then transcription can occur at a faster rate. More often, control is effected at the level of cither transcription or translation, with the former probably being more important for both bacteria and eukaryotes. Transcriptional control in bacteria is particularly effective because of the very short half-life (a few minutes) of mRNA in such cells the half-life is longer in eukaryotes. The prototype for transcriptional control is the lactose operon in E. coli. 

Macrolides, particularly erythromycin and clarithromycin, inhibit CYP3A4. With erythromycin, the inhibition of CYP3A4 is non-competitive due to irreversible binding with the isoenzyme to form an inactive complex. Thus, unlike the case with inhibitors with a short half-life (e.g. cimetidine), the offset of inhibition is slow since new enzyme must be synthesized to replace the inactive complexes. Azithromycin, clarithromycin and erythromycin can prolong the Q-T interval and must not be co-administered with other Q-T-prolonging drugs. 

ALA synthase can be suppressed by glucose. Due to the short half-life of ALA synthase of 70-80 minutes, inhibition or induction of this enzyme very quickly affects haem synthesis. Haem deficiency due to an enzyme defect causes an increase in 5-aminolaevulinic acid. Free haem is either integrated into various apoproteins or it intervenes as a haem repressor with the nuclear gene chain, which leads to the formation of specific mRNA for ALA synthase. Synthesis and consumption of haem are synchronized precisely. The organism produces some 300 mg haem per day, with only 1% being excreted unused in the urine or faeces. (271, 273, 300, 309) (s. p. 34) (s. tab. 3.3)... 

The foregoing section indicates some reasons why peptide-based drugs are needed. If a peptide hormone is not produced in sufficient quantities or is defective in structure, then a replacement is required. Peptides, especially very small molecules, have a very short half life in the body. The reason for this is the ubiquitous occurrence of proteolytic enzymes that effect hydrolysis of peptides to the constituent amino acids. Although longer peptides, especially those with structural features such as disulphide bonds, survive longer in vivo, they are more likely to stimulate the body s immune system to produce antibodies and effect removal of the peptides. This is particularly likely to occur with molecules that differ structurally from the naturally occurring hormones. Thus, treatment of juvenile-onset diabetes mellitus with insulins from animal sources can occasionally stimulate the patient s immune system... 

Glycosylation of insulin has been shown in vitro (D2I). The only other peptide hormone with any significant number of valine and lysine residues is the parathyroid hormone. However, with the short half-life of these hormones in the circulation and tissues, any degree of stable ketoamine formation will be slight. With regard to nonenzymatic glycosylation of enzymes, this has received little attention as yet. 

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