Objective: Understanding the mechanism of action of an enzyme can lead to the construction of hyperactive enzyme variants with a greater catalytic efficiency than the wild type enzyme.
Introduction: 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 have shown that DNase I is also 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 of 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 created more nicks in a shorter period of time would act more efficiently than the wild- type enzyme.
The DNase I variants engineered by the Genentech scientists are listed in the table to the left. Each letter-number-letter combination in the nomenclature of the variant shows the change in an amino acid in the wild-type enzyme. For instance,
“Q9R” means that the glutamine at position 9 in the wild-type DNase I enzyme has been changed to an arginine (find the one-letter amino acid codes).
What structural feature do all of the DNase I variants have in common? Hint: first, explain the meaning of the “abbreviation” column on the table, and then how these changes would arise in the first place. Why did the protein engineering teams think that these changes would improve the catalytic efficiency of DNase I?
![Objective: Understanding the mechanism of action of an enzyme can lead to the
construction of hyperactive enzyme variants with a greater catalytic efficiency than the
wild type enzyme.
Note: Task 33 completes this case study.
Introduction: 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 have shown that DNase I is also 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 of 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 created
more nicks in a shorter period of time
would act more efficiently than the wild-
type enzyme.
Q9R
E13R
T14K
H44K
N74K
T205K
E13R/N74K
Variant
Q9R/E13R/N74K
E13R/N74K/T205
Q9R/E13R/N74K/T205K
E13R/H44K/N74K/T205K
T14K/H44K/N74K/T205K
E13R/T14K/N74K/T205K
Abbreviation
+1
+2
+3
+4
+5
Q9R/E13R/H44K/N74K/T205K
Q9R/E13R/T14K/H44R/N74K/T205K +6
The DNase I variants engineered by the
Genentech scientists are listed in the table
to the left. Each letter-number-letter
combination in the nomenclature of the
variant shows the change in an amino acid
in the wild-type enzyme. For instance,](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F0627210d-c0e3-4a2a-8541-a7399a2e5e96%2F25645a94-5567-489d-b7e7-7d7a21451bc9%2Fk3d89ip_processed.png&w=3840&q=75)
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