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Related Concept Videos

Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Restriction Enzymes01:11

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
Catalytically Perfect Enzymes01:07

Catalytically Perfect Enzymes

The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.
Enzymes02:34

Enzymes

Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Turnover Number and Catalytic Efficiency01:19

Turnover Number and Catalytic Efficiency

The turnover number of an enzyme is the maximum number of substrate molecules it can transform per unit time. Turnover numbers for most enzymes range from 1 to 1000 molecules per second. Catalase has the known highest turnover number, capable of converting up to 2.8×106 molecules of hydrogen peroxide into water and oxygen per second. Lysozyme has the lowest known turnover number of half a molecule per second.
Chymotrypsin is a pancreatic enzyme that breaks down proteins during digestion. The...

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NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases
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Limits to Catalysis by Ribonuclease A.

James E Thompson1, Tatiana G Kutateladze, Michael C Schuster

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

Bioorganic Chemistry
|July 30, 2011
PubMed
Summary

Bovine pancreatic ribonuclease A (RNase A) significantly enhances RNA cleavage by a factor of 3 × 10(11)-fold. This catalysis is primarily limited by desolvation, with the enzyme binding tightly to the transition state.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Bioorganic Chemistry

Background:

  • Bovine pancreatic ribonuclease A (RNase A) is a well-studied enzyme crucial for RNA cleavage.
  • The precise nature of its rate-limiting transition state and catalytic rate enhancement remained largely unknown.
  • Understanding these factors is key to elucidating RNase A's catalytic mechanism.

Purpose of the Study:

  • To investigate the rate-limiting step in RNase A-catalyzed RNA cleavage.
  • To quantify the catalytic rate enhancement and understand the role of the transition state.
  • To determine the influence of solvent effects, such as glycerol and sucrose, on RNase A activity.

Main Methods:

  • Kinetic analysis of wild-type and mutant RNase A using various substrates (UpA, UpOC(6)H(4)-p-NO(2)).
  • Assessing the effect of glycerol and sucrose concentrations on the catalytic efficiency (k(cat)/K(m)).
  • Determining the pH dependence of RNase A activity and comparing catalyzed versus uncatalyzed reaction rates.

Main Results:

  • RNase A's catalytic efficiency (k(cat)/K(m)) for UpA cleavage is inversely related to glycerol concentration, indicating desolvation limitation.
  • Mutant RNase A and a poor substrate showed no glycerol dependence, supporting the desolvation hypothesis.
  • The enzyme achieves a remarkable rate enhancement of 3 × 10(11)-fold for UpA cleavage at pH 6.0 and 25°C.

Conclusions:

  • Catalysis of UpA cleavage by RNase A is limited by desolvation of the transition state.
  • RNase A binds to the transition state with an extremely high affinity (dissociation constant <2 × 10(-15) M).
  • This study provides critical insights into the mechanism of RNase A's potent catalytic activity.