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Related Concept Videos

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...
Reaction Rate02:53

Reaction Rate

The rate of reaction is the change in the amount of a reactant or product per unit time. Reaction rates are therefore determined by measuring the time dependence of some property that can be related to reactant or product amounts. Rates of reactions that consume or produce gaseous substances, for example, are conveniently determined by measuring changes in volume or pressure.
The mathematical representation of the change in the concentration of reactants and products, over time, is the rate...
Multi-Step Reactions02:31

Multi-Step Reactions

Chemical reactions often occur in a stepwise fashion involving two or more distinct reactions taking place in a sequence. A balanced equation indicates the reacting species and the product species, but it reveals no details about how the reaction occurs at the molecular level. The reaction mechanism (or reaction path) provides details regarding the precise, step-by-step process by which a reaction occurs. Each of the steps in a reaction mechanism is called an elementary reaction. These...
Introduction to Enzyme Kinetics01:19

Introduction to Enzyme Kinetics

Enzyme kinetics studies the rates of biochemical reactions. Scientists monitor the reaction rates for a particular enzymatic reaction at various substrate concentrations. Additional trials with inhibitors or other molecules that affect the reaction rate may also be performed.
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Oxidation of Phenols to Quinones01:17

Oxidation of Phenols to Quinones

In the presence of oxidizing agents, phenols are oxidized to quinones. Quinones can be easily reduced back to phenols using mild reducing agents. The electron-donating hydroxyl group enhances the reactivity of the aromatic ring, enabling oxidation of the ring even in the absence of an α hydrogen.
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox property is crucial in...
Phase I Reactions: Reductive Reactions01:27

Phase I Reactions: Reductive Reactions

Phase I biotransformation reductive reactions are chemical processes that modify drugs by introducing or revealing polar functional groups via reduction. Enzymes called reductases catalyze these reactions, playing a pivotal role in drug metabolism by transforming lipophilic drugs into more polar, water-soluble metabolites for easy excretion. An essential type of reductive reaction is the carbonyl group reduction, where aldehydes and ketones are reduced to alcohols. An example is the...

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Related Experiment Video

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Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
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Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXbeta reductase (BVR-B).

O Cunningham1, M G Gore, T J Mantle

  • 1Department of Biochemistry, Trinity College, Dublin 2, Ireland. cunningo@mail.tcd.ie

The Biochemical Journal
|January 6, 2000
PubMed
Summary
This summary is machine-generated.

Biliverin-IXbeta reductase catalyzes flavin reduction via an ordered mechanism, with pyridine nucleotide binding first. The enzyme also exhibits ferric reductase activity, utilizing NAD(P)H and FMN.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Biliverdin-IXbeta reductase exhibits dual activity as both a biliverdin reductase and a flavin reductase.
  • Understanding the enzyme's kinetic mechanisms and substrate interactions is crucial for elucidating its biological roles.

Purpose of the Study:

  • To investigate the initial-rate kinetics of the flavin reductase reaction catalyzed by biliverdin-IXbeta reductase.
  • To characterize the enzyme's interaction with pyridine nucleotides and various flavins.
  • To explore the enzyme's ferric reductase activity and its potential physiological relevance.

Main Methods:

  • Stopped-flow fluorescence quenching to study NADPH binding kinetics.
  • Equilibrium fluorescence quenching to determine binding affinities.
  • Initial-rate kinetic studies under varying pH and buffer conditions.
  • Inhibition studies using lumichrome and mesobiliverdin XIIIalpha.

Main Results:

  • The flavin reductase reaction follows a rapid-equilibrium ordered mechanism at pH 7.5, with pyridine nucleotide binding first.
  • Enzyme-NADPH binding involves an initial encounter complex that isomerizes to a more stable conformation.
  • The enzyme efficiently reduces FMN, FAD, and riboflavin, with distinct K(m) values.
  • Mesobiliverdin XIIIalpha acts as a competitive inhibitor for FMN and shows mixed inhibition with NADPH, suggesting distinct binding sites.
  • Biliverdin-IXbeta reductase also catalyzes ferric iron reduction, requiring NAD(P)H and FMN.

Conclusions:

  • Biliverdin-IXbeta reductase possesses a complex kinetic mechanism and substrate specificity for flavin reduction.
  • The enzyme's ferric reductase activity, coupled with its substrate preference for products of heme cleavage, suggests a role in iron metabolism.
  • Further research is warranted to fully elucidate the physiological significance of these enzymatic activities.