Download Case 31 Hyperactive DNAse I Variants: A Treatment for Cystic

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
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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.
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Questions
1. The DNAse I variants engineered by the Genentech scientists are listed in Table 31.1. (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
+1
H44K
N74K
T205K
E13R/N74K
+2
Q9R/E13R/N74K
+3
E13R/N74K/T205
Q9R/E13R/N74K/T205K
E13R/H44K/N74K/T205K
+4
T14K/H44K/N74K/T205K
E13R/T14K/N74K/T205K
Q9R/E13R/H44K/N74K/T205K
+5
Q9R/E13R/T14K/H44R/N74K/T205K
+6
2. 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?
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3. The DNA hyperchromicity assay was used to measure the KM and vmax values for each variant. The
results are shown in Table 31.2. Explain the significance of the KM and vmax 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.)
KM, μg/mL
DNA
Variant
vmax, A260 units/min/mg
DNAse I
Wild type
1.0
1.0
Q9R
1.1
2.8
E13R
0.23
1.5
T14K
0.43
1.1
H44K
0.43
1.1
N74K
0.77
3.6
T205K
0.42
2.1
E13R/N74K
0.20
5.3
Q9R/E13R/N74K
0.20
7.0
E13R/N74K/T205
0.18
7.7
Q9R/E13R/N74K/T205K
0.09
2.8
E13R/H44K/N74K/T205K
0.17
6.4
T14K/H44K/N74K/T205K
0.18
7.7
E13R/T14K/N74K/T205K
0.37
3.5
Q9R/E13R/H44K/N74K/T205K
0.11
2.4
Q9R/E13R/T14K/H44R/N74K/T205K
0.20
2.5
4. 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 31.1. 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
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electrophoresis. The results are shown in Figure 31.2. 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
+3
+4
+6
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
wild type DNAse I, +3 is 13R74K205K, +4 is 13R14K74K205K and +6
is 9R13R14K44R74K205K. (Based on Pan and Lazarus, 1998.)
5. The
investigators next
tested the
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 31.3. 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.)
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Relative nicking activity (as compared to wild type)
Low DNA conc
High DNA conc
Low mwt
High mwt
Low mwt
High mwt
Wild type
1
1
1
1
N74K
26
E13R/N74K
211
E13R/N74K/T205K
7
1.3
E13R/T14K/N74K/T205 K
7
0.7
10.4
31
24.3
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6. 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. 11701-11708.
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