top of page

Enzymes

image.png

Roles of Enzymes

definition: highly specialized proteins that catalyze reactions in biological systems

  • decreases activation energy for a specific chemical reaction

  • are specific for one set of substrates

  • are not changed nor consumed in the reaction

nomenclature

  • usually ends with -ase

  • attached to substrate (urease) or description of action performed (lactate dehydrogenase)

  • sometimes have original names (trypsin)

image.png

6 Major classes of enzymes: 

  • ​Oxidoreductases

    • catalyze REDOX reactions​

    • ex. lactate dehydrogenase

  • Transferases

    • catalyze transfer of C,N or P containing groups​

    • ex. serine hydroxylmethyl transferase

  • Hydrolases

    • catalyze cleavage of bonds by addition of water​

    • ex. urease

  • Lyases

    • catalyze cleavage of C-C, C-S, and some C-N bonds​

    • ex. pyruvate decarboxylase

  • Isomerases

    • catalyze racemization of isomers​

    • ex. methylmalonyl CoA mutase

  • Ligases

    • catalyze formation of bonds between C, O, S and N coupled to hydrolysis of high energy phosphates​

    • ex. pyruvate carboxylase

Molecular Structure

  • Enzyme molecular structure

    • —Active site

      • —Part of protein that where reactants come together

      • —That way doesn’t rely on chance encounters

      • —Arrangement of atoms into a 3-dimensional cleft or crevice

        • —Substrate fits into the crevice—in most enzymes it’s an exact fit

          • “Lock and key”

        • —Some enzymes: induced fit active site = active site is molded after the substrate engages

  • Enzymes are highly specific for their substrate

  • The reactant an enzyme acts on is the substrate

    • — enzymes bind to substrates

      • — enzyme-substrate complex

  • The reaction catalyzed by each enzyme is very specific.

    • —results from its 3-dimensional shape, a consequence of its amino acid sequence.

  • The active site of an enzyme is typically a pocket or groove on the surface of the protein

    • —As the substrate enters the active site, steric interactions between the chemical groups on the substrate and the R groups of amino acids of the protein cause the enzyme to change shape

  • —This brings the chemical groups of the active site into position to catalyze the reaction.

image.png

k1

k2

k3

k4

Induced Fit Model

image.png

​

  1. Active site approximately fits substrates (e.g., all proteins) 

  2. As substrate(s) begins to bind, conformation change in enzyme allows for better fit

  3. Reaction takes place

study question: 

  • how does the induced fit model explain enzyme-substrate specificity compared to the older lock and key model?

image.png

How do Enzymes Work?

  • —Enzymes don’t work alone

    • —Use coenzymes and cofactors

      • Cofactor (inorganic) e.g., Mg, Fe

        • not protein based​

        • hold normal enzyme shape

        • allows substrate to bind to active site

      • Coenzyme (organic) e.g., vitamin based NAD, FAD

        • vitamin derived​

        • does not directly interact with enzyme, is involved in reaction

        • used for multiple reactions​

        • ​transfer small chemical groups from one reaction to another

  • —Nomenclature

    • —Holoenzyme = protein + cofactor (coenzyme)

    • —Apoenzyme = protein alone (inactive)

  • This is the role of many vitamins in metabolism

study questions: 

  • what 3 coenzymes are important in energy metabolism?

  • what is the main difference between cofactor and coenzyme?

image.png

Factors Affecting Enzymatic Rates

1. Catalytic Rate

  • Amount of product produced per unit time

  • Assumption: enzyme active site always occupied by substrate

2. Concentration

  • —Greater enzyme concentration corresponds to greater reaction rate based on law of mass action

  • —Greater substrate concentration – shows saturation (asymptotic response)

V

image.png

study questions: 

  • how does substrate concentration affect enzymatic rate?

  • what occurs when enzymes are fully saturated?

  • number of collisions is dependent on the number of substrate molecules

  • reaction rate (V) is proportional to [S]

    • low [S] = first order reaction rate (linear)

  • when there is a large number of substrates, all enzymes cannot react at once

    • reaction rate increases and then plateaus at maximum rate/velocity (Vmax)​

    • when product forms, more enzymes become available for substrate to react, creating equilibrium

    • high [S] = zero order reaction rate

3. Ligand-Protein Interactions (Affinity)

  • ligand - molecules that bind proteins by weak interactions

  • only hydrogen and ionic bonds, not covalent

  • affinity is the measure of strength of binding between substrate and enzyme

4. Acidity (pH)

5. Temperature

image.png

study question: 

  • describe how affinity impacts reaction rate

image.png

Km, Vmax and Reaction Rates

image.png
image.png
image.png
What is Km?

  • Km is a substrate concentration

    • Units must be in concentration

    • A “constant” or defining feature of enzyme for a given set of conditions

  • Km is a property of every enzyme molecule…it does NOT depend upon the enzyme concentration.  Thus it is an “intensive” constant, in contrast to Vmax.

  • Km is inversely related to the affinity of an enzyme for its substrate

    • the higher the affinity the lower the Km.

image.png

study question: 

  • describe the relationship between Km and Vmax. how do they both impact enzyme rates?

  • lower Km means the enzyme has a higher affinity to the substrate

  • higher Km means the enzyme has a lower affinity to the substrate

  • Michaelis - Menton

    • Vi (initial velocity) = Vmax[S] / Km + [S]

    • Km = [S] at 1/2 Vmax

  • when [S] < Km

    • Lots of enzymes active sites are available

    • As ­ [S], has strong effect on rate

    • First order kinetics--proportional to amount added

  • when [S] > Km

    • Because almost all active sites are occupied (saturated) there is limited amount of enzyme

    • More substrate does not affect rate more after reaction gets going

    • Zero order kinetics or independent of [S]

image.png
image.png

refer back to image below

In this scheme: 

Km = (k2 + k3) / k1

image.png

k1

k2

k3

k4

  • Michaelis-Menten equation can be rearranged to linear form: Lineweaver-Burke

    • ​1/v = (Km/Vmax)(1/[S]) + 1/Vmax​

image.png

Enzyme Inhibitors 

  • Inhibitors are small molecules that bind to an enzyme and reduce its catalytic ability.

  • There are two major classes of inhibitors:

    •  Reversible inhibitors can dissociate from the enzyme once they are bound

      • competitive, competitive, uncompetitive ​

    • Irreversible inhibitors can not dissociate from the enzyme.

Competitive

Competitive inhibitors react only with the free enzyme, often by binding to the active site…thus they “compete” with substrate for binding. 

image.png
image.png

study question: 

  • explain the differences between the 3 types of reversible inhibitors. 

image.png
image.png
image.png
Screenshot 2024-07-11 at 4.07.16 PM.png

  • Inhibitor reversibly binds to the active site (where substrate would bind), therefore completing with substrate

    • reversed by increasing [S]

    • at sufficient [S], can still reach Vmax

    • increases Km for substrate

  • more substrate is needed to achieve 1/2 Vmax

  • the lineweaver-burke equation that describes the kinetics in the presence of a competitive inhibitor are altered by the term (1 + [I]/KI) as follows:​​

image.png
image.png
Non- Competitive

Non-competitive inhibitors react with the free enzyme and the enzyme substrate complex.  Thus they usually bind to a site on the enzyme surface away from the active site.

  • decreased Vmax

  • occurs when inhibitor and substrate bind to different sites on the enzyme

  • can bind either the free enzyme of the ES complex and prevent reaction

  • cannot be overcome by increasing [S]

  • does not interfere with S binding to enzyme, no effect on Km

  • Vmax cannot be attained in the presence of a NC inhibitor

  • The lineweaver-burke equation that describes the kinetics in the presence of a non-competitive inhibitor are altered by the term (1 + [I]/KI) as follows:

image.png
image.png
image.png
image.png
Screenshot 2024-07-11 at 4.14.41 PM.png
Uncompetitive

Uncompetitive inhibitors react only with the substrate bound form of the enzyme

image.png

The lineweaver-burke equation that describes the kinetics in the presence of an uncompetitive inhibitor are altered by the term (1 + [I]/KI) as follows:

image.png
image.png
image.png
Irreversible

  • Irreversible inhibitors inactivate enzymes by covalently binding them.

  • The kinetics of an irreversible inhibitor are quite easy to interpret: addition of inhibitor continually lowers Vmax until all enzyme molecules have reacted stoichiometrically with the inhibitor, at which point there will be no active enzyme molecules left.

  • Some irreversible inhibitors, called suicide substrates, look like the natural substrate but covalently attach to the enzyme at some point during binding and/or catalysis…high concentrations of substrate can temporarily protect the enzyme from such inhibitors.

Regulation of Enzyme Activity

1. Allosteric regulation​​

  • ​an enzyme has two binding sites: active and regulatory

  • modulator molecule binds to regulatory site which changes shape and activity of enzyme

  • can increase or decrease activity

  • generally alters affinity and catalytic rate

2. Covalent regulation

3. Feedback inhibition

4. Feedforward activation

©2023 by Syracuse University Dr.Margaret Voss

bottom of page