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Related Concept Videos

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...
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.
Introduction to Mechanisms of Enzyme Catalysis01:13

Introduction to Mechanisms of Enzyme Catalysis

For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild...
Introduction to Mechanisms of Enzyme Catalysis01:13

Introduction to Mechanisms of Enzyme Catalysis

For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild...
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...
Enzyme Kinetics01:19

Enzyme Kinetics

Enzymes speed up reactions by lowering the activation energy of the reactants. The speed at which the enzyme turns reactants into products is called the rate of reaction. Several factors impact the rate of reaction, including the number of available reactants. Enzyme kinetics is the study of how an enzyme changes the rate of a reaction.
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...

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Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota
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Structure-function relationships of a catalytically efficient beta-D-xylosidase.

Douglas B Jordan1, Xin-Liang Li, Christopher A Dunlap

  • 1US Department of Agriculture, Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, Peoria, IL 61604, USA. jordand@ncaur.usda.gov

Applied Biochemistry and Biotechnology
|July 13, 2007
PubMed
Summary

Selenomonas ruminantium beta-D-xylosidase efficiently hydrolyzes xylooligosaccharides via an inversion mechanism. Key residues D14 and E186 act as general base and acid, respectively, influencing substrate binding and catalysis.

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Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition
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Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Published on: October 3, 2018

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Beta-D-xylosidases are crucial enzymes for biomass degradation.
  • Understanding their catalytic mechanisms is vital for industrial applications.
  • Selenomonas ruminantium beta-D-xylosidase (GH43) is a promising candidate for efficient hydrolysis.

Purpose of the Study:

  • To elucidate the catalytic mechanism and substrate specificity of beta-D-xylosidase from Selenomonas ruminantium.
  • To identify key active site residues involved in hydrolysis.
  • To investigate the enzyme's interaction with different xylooligosaccharide substrates.

Main Methods:

  • Enzyme kinetics studies using various substrates, including 4-nitrophenyl-beta-D-xylopyranoside (4NPX) and xylooligosaccharides.
  • 1H nuclear magnetic resonance (NMR) spectroscopy to determine the reaction mechanism.
  • Site-directed mutagenesis (D14A, E186A) to probe the role of specific residues.
  • Homology modeling to visualize enzyme-substrate interactions.

Main Results:

  • The enzyme exhibits high catalytic efficiency (kcat, kcat/Km) for 1,4-beta-D-xylooligosaccharides, acting via an inversion mechanism.
  • Catalytic efficiency decreases for longer oligosaccharides, highlighting the importance of subsites -1 and +1.
  • Mutagenesis studies confirm D14 and E186 as essential catalytic residues (general base and acid, respectively).
  • pH-dependent kinetics reveal pKa values for D14 (5.0) and E186 (7.0), influencing substrate binding and catalysis.

Conclusions:

  • Selenomonas ruminantium beta-D-xylosidase is a highly effective catalyst for xylooligosaccharide hydrolysis.
  • The enzyme utilizes an inversion mechanism, with D14 and E186 playing critical roles in catalysis.
  • Substrate binding and activity are influenced by pH and the enzyme's active site architecture.