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BMED 3510
Metabolic Systems
Book Chapter 8
What is Metabolism?
Etymology: Greek “meta · ballein” ~ to throw about, to change
Metabolism is the set of life-sustaining chemical transformations within
cells of living organisms.
Metabolites are the intermediates and products of metabolism. The
term ''metabolite'' is usually restricted to small molecules.
A primary metabolite is directly involved in processes of normal
growth, development, and reproduction (e.g. glucose and pyruvate).
A secondary metabolite is not directly involved in those processes, but
usually has an important ecological function. Examples include
antibiotics and pigments.
http://www.news-medical.net/; en.wikipedia.org
What is Metabolomics?
Metabolomics is the scientific study of biochemical systems
involving large numbers of metabolites at the same time.
Metabolomics is the systematic study of the unique chemical fingerprints
that specific cellular processes leave behind.
Currently there are ≈42,000 metabolites in HMDB2, of which:
22,000 are associated with a proteins (enzyme & transporters)
5,000 have been quantified.
en.wikipedia.org & www.hmdq.ca
Problem Assessing Metabolites
The metabolome unlike the genome or the proteome is
chemically very diverse.
Metabolites can be water-soluble, lipid-soluble, gases,
organic/inorganic, positively/negatively charged (sol)…
Wide concentration range: from the molar range down to nothing.
Present in several compartments (cytosol, mitochondrial, ER, ect).
Half-lives of metabolites are extremely variable, with
some metabolites being very short-lived inside cells
Assessing Metabolites
Nature Protocols 6, 1241–1249 (2011)
Quenching in Cold Methanol
Nature Protocols 6, 1241–1249 (2011)
Detection
Http://www.bruker.com
Metabolic networks
Http://www.infohow.org
Thermodynamics
• Spontaneous reactions only
occur between high and low
energy metabolites.
(Reactions w/ negative ΔG)
• Achieved by:
Coupled reactions.
Prentice Hall c2002
What are Enzymes?
Proteins that catalyze (bio)chemical reactions
Why do we Have Enzymes?
• Higher reaction rates
•
Most reactions don’t even occur spontaneously.
• Greater reaction specificity
•
•
•
Absolute
Relative (group)
Stereospecificity (Stereoisomers)
• Milder reaction conditions
• Capacity for regulation
COO
-
COO
• Metabolites have
many potential
pathways of
conversion or
decomposition
NH2
O
OH
COO
OH
COO
Chorismate
mutase
COO
OOC
O
NH2
-
-
O
COO
COO
OH
-
• Enzymes allow
substrate to be
channeled into the
product of highest
demand
In the Absence of an Enzyme
S
P
In the Presence of an Enzyme
S
ES
EP
P
How is ∆G Lowered?
Catalytic Cycle of an Enzyme
1 Substrates enter active site; enzyme
changes shape so its active site
embraces the substrates (induced fit).
Substrates
Enzyme-substrate
complex
6 Active site
Is available for
two new substrate
Mole.
Enzyme
5 Products are
Released.
Figure 8.17
Products
2 Substrates held in
active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.
3 Active site (and R groups of
its amino acids) can lower EA
and speed up a reaction by
• acting as a template for
substrate orientation,
• stressing the substrates
and stabilizing the
transition state,
• providing a favorable
microenvironment,
• participating directly in the
catalytic reaction.
4 Substrates are
Converted into
Products.
Enzyme Kinetics
•
•
Thermodynamics: Energy and possibility of a reaction
Kinetics: Speed of a reaction
•
•
Kinetics is the study of the rate at which compounds react
Rate of enzymatic reaction is affected by
– Enzyme
– Substrate & Products
– Effectors (Inhibitors & Activators)
– Environmental conditions (Temperature, pH)
Measuring Enzyme Kinetics
Kinetic Properties of Enzymes
Study of the effect of substrate concentration on the rate of reaction
Henri-Michaelis-Menten Kinetics
Adian Brown (1902)
Victor Henri (1903)
Archibald Vivian Hill (1910)
Michaelis & Menten (1913)
Briggs & Aldane (1925)
E
+
S
k1
k-1
E S
k2
E
S  k1  S  E  k1  ( ES )
( E S )  k1  S  E  (k1  k2 )  ( ES )
P  k 2  ( ES )
+
P
Henri-Michaelis-Menten Kinetics
Assumptions:
S  k1  S  E  k1  ( ES )
( E S )  k1  S  E  (k1  k2 )  ( ES )
P  k  ( ES )
Concentrations
2
• Reaction mechanism
• Homogeneous (well stirred)
• k1, k-1 >> k2
• S >> ET (ET=E+ES)
P
• Quasi-Steady-State Assumption:
S

( ES )  0
ES
Time
Henri-Michaelis-Menten Kinetics
( E S )  k1  S  E  (k1  k2 )  ( ES )  0
k1  S  E  (k1  k2 )  ( ES )
( ES ) 
k1
S E
S E 
k 1  k 2
Km
K m  ( ES )  S  ( ET  ES )
( ES ) 
S  ET
Km  S
S  k 2  ET S  Vmax
v  P  k 2  ( ES ) 

Km  S
Km  S
Km 
k 1  k 2
k1
ET  E  ES
P  k 2  ( ES )
Vmax  k2  ET
Henri-Michaelis-Menten Kinetics
Reaction speed
Vmax
vP
Vmax S
vp 
KM  S
Vmax/2
KM
[S]
Henri-Michaelis-Menten Kinetics
• KM is the apparent dissociation constant of the ES complex
and measures the enzyme’s affinity for the substrate.
- KM values of enzymes differ widely.
- KM provides approximation of substrate concentration in vivo.
- For most enzymes, KM lies between 10-1 and 10-7M.
- High KM indicates weak binding.
- Low KM indicates strong binding.
• Vmax = kcat ET
• kcat (k2) is the “turnover number”: measures how many substrate
molecules one enzyme molecule converts per unit of time.
• ET is the total enzyme concentration
- Most enzymes are not saturated with substrate
- kcat/KM can be used as a measure of catalytic efficiency.
- Diffusion limits the catalytic efficiency. Why? kcat/KM < K1
Hill Kinetics
Reactions catalyzed by enzymes with n subunits
Reaction speed
Vmax
vP
n
Vmax S
vp  n
KM  S n
Vmax/2
KM
[S]
Common Enzymatic Mechanisms
Enzyme Inhibition
Inhibitors are compounds that decrease the enzyme activity
• Irreversible inhibitors (inactivators) react with the enzyme
- one inhibitor molecule can permanently shut off one enzyme molecule
- they are often powerful toxins but also may be used as drugs
• Reversible inhibitors bind to, and can dissociate from the enzyme
- they may be structural analogs of substrates or products
- they are often used as drugs to slow down a specific enzyme
• Competitive inhibition: the inhibitor competes with the substrate for the active site.
-Can be overcome by a sufficiently high concentration of substrate.
-Inhibitor increase the Km value.
• Allosteric inhibition: the inhibitor binds to a different binding site.
Competitive Inhibition
Vmax S 
v
S   K m 1  I  
 KI 
KM is modulated
Uncompetitive & Noncompetitive
Effects of Inhibitors
Effects of Inhibitors
http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olinhibition.html
http://www.as.wvu.edu/~rbrundage/lecture2b/img011.gif
Databases: BRENDA
Databases: KEGG
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Pathway Regulation
Negative feedback
• Product of a pathway controls the rate of synthesis by inhibiting an early
step, usually the first “committed” step (unique to the pathway).
Positive feedforward
• Metabolite early in the pathway activates an enzyme further down the
pathway
Glycolysis in L. lactis
A.R. Neves et al. FEMS Microbiol. Rev. 29 (2005) 531–554
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Amino Acid Biosynthesis
http://www.uky.edu/~dhild/biochem/24/lect24.html
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Reversible Covalent Modification
Reversible Covalent Modification
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Insulin-Induced Glucose Uptake
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Multifunctional Enzymes
Metabolic Regulation
How is metabolism regulated?
• Enzymatic regulation: activation or Inhibition (comp., uncomp.)
• Pathway regulation:
- Negative feedback
- Positive feedforward
• Isoenzymes, two or more proteins that catalyze the same reaction
- Different kinetics, regulatory properties and cellular distribution
- Coded by different genes or by alternative splicing
• Reversible covalent modification
• Compartmentalization
• Multienzyme complexes and multifunctional enzymes
- Metabolite channeling - “channeling” of reactants between active sites
• Transcription, translation, mRNA and protein degradation
Summary
Advantages of having enzyme catalyzed reactions
How do enzymes operate?
Michaelis-Menten rate law (approximation)
Effect of different types of inhibition
Metabolic regulation
many modes, operating at different time scales