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Related Experiment Videos

Structural determinants of enzymatic processivity

S B delCardayré1, R T Raines

  • 1Department of Biochemistry, University of Wisconsin-Madison 53706-1569.

Biochemistry
|May 24, 1994
PubMed
Summary
This summary is machine-generated.

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Protein engineering·2001

Researchers modified the bovine pancreatic ribonuclease A (RNase A) enzyme to alter its substrate specificity. Mutants showed increased specificity for purine bases, demonstrating substrate binding can be separated from catalytic turnover.

Area of Science:

  • Enzymology
  • Protein Engineering
  • Biochemistry

Background:

  • Bovine pancreatic ribonuclease A (RNase A) is a nonprocessive endoribonuclease.
  • RNase A utilizes three subsites (B1, B2, B3) with specific base preferences (pyrimidine, adenine, purine) for RNA binding.
  • The B1 subsite typically binds pyrimidine bases.

Purpose of the Study:

  • To investigate the role of specific amino acid residues in RNase A's substrate specificity.
  • To engineer RNase A mutants with altered catalytic properties and purine/pyrimidine specificity.
  • To explore the relationship between substrate binding and catalytic turnover in nonprocessive enzymes.

Main Methods:

  • Site-directed mutagenesis was used to substitute residues Thr45 and Phe120 in the B1 subsite of RNase A.

Related Experiment Videos

  • Mutant enzymes were screened for altered RNA cleavage specificity, particularly for purine residues.
  • Enzyme activity and processivity on various polyribonucleotides (poly(A), poly(C), poly(U)) were analyzed.

    • Main Results:

    • Mutant enzymes, specifically Ala45 and Gly45, exhibited efficient cleavage of poly(A), poly(C), and poly(U).
    • These mutants displayed a 10^3- to 10^5-fold increase in purine/pyrimidine specificity compared to wild-type RNase A.
    • The Ala45 and Gly45 mutants demonstrated processive cleavage of poly(A), uncoupling substrate binding from turnover.

    Conclusions:

    • Substrate binding can be decoupled from substrate turnover in RNase A catalysis through specific mutations.
    • A new model for processive enzymes is proposed, suggesting subsites specific for repeating motifs within polymeric substrates.
    • Processive enzymes may exhibit tighter binding to repeating substrate motifs compared to non-repeating ones.