Prerequisites Enzyme kinetics and inhibition. DNA structure. The hyperchromic effect. The properties of supercoiled DNA.
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1 Case 31 Hyperactive DNAse I Variants: A Treatment for Cystic Fibrosis Focus concept Understanding the mechanism of action of an enzyme can lead to the construction of hyperactive variant enzymes with a greater catalytic efficiency than the wild type enzyme. Prerequisites Enzyme kinetics and inhibition. DNA structure. The hyperchromic effect. The properties of supercoiled DNA. Background The enzyme deoxyribonuclease I (DNAse I) is an endonuclease that hydrolyzes the phosphodiester bonds of the double-stranded DNA backbone to yield small oligonucleotide fragments. DNAse I is used therapeutically to treat patients with cystic fibrosis (CF). The DNAse I enzyme is inhaled into the lungs where it then acts upon the DNA contained in the viscous sputum secreted by the lungs in these patients. Hydrolysis of high molecular weight DNA to low molecular weight DNA in the sputum decreases its viscosity and improves lung function. Animal studies also have shown that DNAse I is effective in treating the autoimmune disease systemic lupus erythematosus (SLE). In this disease, the DNA secreted into the serum provokes an immune response. DNAse I prevents the immune response by degrading the DNA to smaller fragments that are not recognized by the immune system. Genentech, Inc., the company that produces the recombinant DNAse I, was interested in improving the efficiency of DNAse I so that less drug would be needed to achieve the same results. Scientists in the protein engineering lab constructed hyperactive variants at DNAse I which actually worked better than the wild-type enzyme. DNAse I acts by processively nicking the phosphodiester backbone, so the scientists reasoned that a variant that could create more nicks in a shorter period of time would act more efficiently than the wild-type enzyme. In this case, we will examine the engineered hyperactive variants and use the results to make some conclusions about the mechanism of DNAse I. 1
2 Questions 1. The DNAse I variants engineered by the Genentech scientists are listed in Table (A note on nomenclature: Q9R mean that the glutamine at position 9 in the wild type DNAse I enzyme has been changed to an arginine.) a. What structural feature do all of the DNAse I variants have in common? Explain the meaning of the abbreviation in the table. b. Why do you suppose that the protein engineers thought that these changes would improve the catalytic efficiency of DNAse I? Table 31.1: DNAse I variants Variant Abbreviation Q9R E13R T14K H44K +1 N74K T205K E13R/N74K +2 Q9R/E13R/N74K +3 E13R/N74K/T205 Q9R/E13R/N74K/T205K E13R/H44K/N74K/T205K T14K/H44K/N74K/T205K +4 E13R/T14K/N74K/T205K Q9R/E13R/H44K/N74K/T205K +5 Q9R/E13R/T14K/H44R/N74K/T205K The enzymatic activity of the DNAse I variants was tested using a DNA hyperchromicity assay. The absorbance of a solution of intact DNA was measured at 260 nm. Then the enzyme was added, and the solution was monitored for an increase in absorbance. (The increase in absorbance at 260 nm is referred to as the hyperchromic effect.) Why was a hyperchromicity assay effective in assessing the activity of the DNAse I variants? 2
3 3. The DNA hyperchromicity assay was used to measure the K M and v max values for each variant. The results are shown in Table Explain the significance of the K M and v max values. What effect has the amino acid change(s) had on the activity of the enzyme variants as compared to the wild type? Table 31.2: DNAse I variants. (Based on Pan and Lazarus, 1998.) Variant K M, μg/ml DNA v max, A 260 units/min/mg DNAse I Wild type Q9R E13R T14K H44K N74K T205K E13R/N74K Q9R/E13R/N74K E13R/N74K/T Q9R/E13R/N74K/T205K E13R/H44K/N74K/T205K T14K/H44K/N74K/T205K E13R/T14K/N74K/T205K Q9R/E13R/H44K/N74K/T205K Q9R/E13R/T14K/H44R/N74K/T205K Next, the protein engineers wished to characterize the DNAse I variants in terms of their ability to cut or nick DNA. A cut refers to the hydrolysis of phosphodiester bonds on both strands, whereas a nick is the hydrolysis of just one strand. This was assessed by using the circular plasmid pbr322. The plasmid is the most stable in the supercoiled form. If the phosphodiester backbone is nicked on one strand, the plasmid forms a relaxed circle, but if the backbone is cut on both strands, the circle linearizes, as shown in Figure Supercoiled, relaxed circular and linear DNA can be detected by differential migration through agarose gels. In a series of experiments, pbr322 substrate was incubated with DNAse I for 45 minutes, then the products were analyzed by agarose gel 3
4 electrophoresis. The results are shown in Figure Describe the results for each lane. Compare the selected variants with the wild type DNAse I with regard to their ability to cut or nick the DNA. Figure 31.1: Conversion of supercoiled DNA to relaxed and linear DNA via phosphodiester bond hydrolysis. C WT Relaxed circle Linear Supercoiled Figure 31.2: Supercoiled plasmid DNA digestion by DNAse I variants and analysis by agarose gel electrophoresis. C is the control, WT is 5. The wild type DNAse I, +3 is 13R74K205K, +4 is 13R14K74K205K and +6 investigators next tested the is 9R13R14K44R74K205K. (Based on Pan and Lazarus, 1998.) DNAse I variants enzymatic ability with high and low molecular weight DNA, in high and low concentrations. High molecular weight DNA is present in the lung secretions of CF patients in fairly high concentrations, but the DNA present in the serum of mice with SLE is present in one-tenth the concentration. The data are shown in Table What is your interpretation of these data? Table 31.3: Dependence of DNA nicking activity by DNAse I variants on DNA length and concentration. (Based on Pan and Lazarus, 1998.) 4
5 Relative nicking activity (as compared to wild type) Low DNA conc High DNA conc Low mwt High mwt Low mwt High mwt Wild type N74K E13R/N74K E13R/N74K/T205K E13R/T14K/N74K/T205 K Use the experimental evidence presented here to compare the mechanism of the wild type DNAse I with the variant DNAse I enzymes. Reference Pan, C. Q., and Lazarus, R. A. (1998) J. Biol. Chem. 273, pp
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