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?
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