(JUNE 2011)
18. A researcher has isolated a restriction endonuclease that cleaves at only one specific 10
base pair site.
A) Would this enzyme be useful in protecting cells from viral infections, given that a typical viral genome is 5 x 10⁴ base pairs long?
B) Restriction endonucleases are slow enzymes with turnover number of 1 s^-1. Suppose the
isolated endonuclease was faster with turnover numbers similar to those for carbonic anhydrase (10⁶ s^-1), would this increased rate be beneficial to host cells, assuming that the fast enzymes have similar levels of specificity?
The correct combination of answer is
(1) (A) : No (B) : Yes                                      (2) (A) : No (B) : No
(3) (A) : Yes (B) : No                                      (4) (A) : Yes (B) : Yes

The correct answer is (4) (A) : Yes (B) : Yes.

Introduction

Restriction endonucleases are bacterial enzymes that defend host cells against viral DNA by cleaving specific DNA sequences. This defense mechanism depends on the frequency of recognition sites in viral genomes and the catalytic efficiency of these enzymes. This article addresses two key questions: the usefulness of a restriction enzyme that recognizes a single 10 base pair site in a viral genome, and whether a higher turnover number (enzyme speed) enhances host cell protection, while maintaining enzyme specificity.

Part A: Usefulness of Enzyme Recognizing Only One Specific 10 bp Site

• The viral genome length: approximately 5×1045×104 base pairs.

• The restriction enzyme recognizes one specific 10 bp sequence.

Probability and Frequency of Recognition Site

• Random occurrence frequency of 1 specific 10 bp sequence:

(1/4)10=11,048,576≈10−6(1/4)10=1,048,5761≈10−6

• For a viral genome of 5×1045×104 bp, expected number of recognition sites:

5×104×10−6=0.055×104×10−6=0.05

• Thus, the specific recognition site may be present less than once, or very rarely, in viral genomes of this size.

Conclusion

• The enzyme may not recognize nor cleave the viral DNA effectively due to the extremely low probability of the 10 bp site occurring.

• Hence, it is not useful for protecting cells from viral infection when the target site is unique and rare.

Part B: Effect of Increased Turnover Number Comparable to Carbonic Anhydrase

• Restriction endonucleases typically have turnover numbers (kcatkcat) around 1 s−11s−1, relatively slow.

• Carbonic anhydrase has an exceptionally high turnover number (~106 s−1106s−1).

• Suppose the restriction enzyme has turnover kinetics similar to carbonic anhydrase, but specificity remains constant.

Benefits of Increased Turnover Number

• Faster cleavage of viral DNA upon recognition.

• More enzymes effectively degrading invading viral genomes in shorter time frames.

• Enhanced protection since the increased enzyme velocity lowers viral replication before it becomes harmful.

Assumptions

• Specificity is equally high to avoid nonspecific cleavage of host DNA.

• High turnover enhances efficiency without introducing off-target effects.

Conclusion

• Increased enzyme speed would benefit host cells, enabling rapid neutralization of viruses.

Final Correct Combination

• (A) No: The enzyme recognizing a single rare site is insufficient for effective protection.

• (B) Yes: Higher turnover number improves defensive efficiency if specificity remains.

So, the correct answer is (1) (A) : No (B) : Yes.

Biological Significance

• Restriction-modification systems are a form of primitive bacterial immune defense primarily against bacteriophages.

• Enzymes must balance specificity and catalytic efficiency.

• High rate enzymes with maintained specificity can improve host survival during infections.

• Viral genomes often evolve to evade restriction sites or produce methylation to prevent cleavage.

Summary Table

Conclusion

A restriction endonuclease recognizing only one rare site in a viral genome is unlikely to effectively protect bacterial cells. However, if such an enzyme exhibits a high turnover number comparable to carbonic anhydrase while maintaining substrate specificity, it would enhance protection by rapidly cleaving viral DNA. This understanding reveals the delicate balance bacteria maintain in defensive enzyme evolution and function, providing insights for biotechnology and molecular biology applications