Triose phosphate isomerase expression

A quantitative expression developed by Albery and Knowles to describe the effectiveness of a catalyst in accelerating a chemical reaction. The function, which depends on magnitude of the rate constants describing individual steps in the reaction, reaches a limiting value of unity when the reaction rate is controlled by diffusion. For the interconversion of dihydroxacetone phosphate and glyceraldehyde 3-phosphate, the efficiency function equals 2.5 x 10 for a simple carboxylate catalyst in a nonenzymic process and 0.6 for the enzyme-catalyzed process. Albery and Knowles suggest that evolution has produced a nearly perfect catalyst in the form of triose-phosphate isomerase. See Reaction Coordinate Diagram... 

Some caution should be exercised however when working with synthetic enzymes. A similarly designed and expressed de novo enzyme to mimic the naturally occurring triose phosphate isomerase was reported in 2004 only for the papers to be retracted when it appeared that contamination by unmodified Escherichia coli had been responsible for the observed enzymic activity. For this reason it seems preferential to target reactions that have no known natural catalysts. 

A number of other enzymopathic substances (e.g., pyruvate kinase. Chapter 13 and pyrimidine-5 -nucleotidase. Chapter 27), abnormal hemoglobins (Chapter 28), and abnormalities of the erythrocyte cytoskeleton (Chapter 10) may cause hemolytic anemia. Because many enzymes in the red cell are identical to those in other tissues, defects in these enzymes may have pleiotropic effects. Thus, in addition to hemolytic anemia, triose phosphate isomerase deficiency causes severe neuromuscular disease, and phospho-fructokinase deficiency causes a muscle glycogen storage disease (Chapter 13). Mutations that result in decreased enzyme stability are usually most strongly expressed in erythrocytes because of their inability to synthesize proteins. 

Fig. 13.1 S. cerevisiae expression vector used for production of recombinant insulin. The S. cerevi-siae/E. coli shuttle vector is composed of the following genetic units 1) The transcription promoter and terminator of the S. cerevisiae TPIl (triose phosphate isomerase) gene flanking the DNA encoding the leader-insulin precursor. 2) The TPIl gene from Schizosaccharomyces pombe (TPIlp)...

Deletion mutants, in which the bla signal sequence is almost completely removed, e.g., Met-Ser-(263 amino acid /8-lactamase) or Met-Arg-Ser-(263 amino acid /8-lactamase), produce but do not secrete the /8-lactamase (62). Note The extra 2 or 3 amino acids at the N terminus do not affect the catalytic activity of the enzyme.) The fusion protein expressed from the bla signal codons fused to an intracellular protein (e.g., triose-phosphate isomerase) gene is neither secreted into the periplasm of E. colt nor proteolytically processed. 

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