Transcript ENZYMES Medical Biochemistry, Lecture 23 Lecture 23, Outline • • • • • • Definition of enzyme terms and nomenclature Description of general properties of enzymes Binding energy and transition.

ENZYMES
Medical Biochemistry, Lecture 23
Lecture 23, Outline
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Definition of enzyme terms and nomenclature
Description of general properties of enzymes
Binding energy and transition states
Catalytic mechanisms and functional groups
In book, Chapters 8,10; Ignore pp 78-80
Recommended supplement for lectures 23-25:
UNDERSTAND Biochemistry CD
Enzyme Catalysis Overview
Enzyme Nomenclature
• active site - a region of an enzyme comprised of
different amino acids where catalysis occurs
(determined by the tertiary and quaternary structure
of each enzyme)
• substrate - the molecule being utilized and/or
modified by a particular enzyme at its active site
• co-factor - organic or inorganic molecules that are
required by some enzymes for activity. These
include Mg2+, Fe2+, Zn2+ and larger molecules
termed co-enzymes like nicotinamide adenine
dinucleotide (NAD+), coenzyme A, and many
vitamins.
Enzyme Nomenclature (cont)
• prosthetic group - a metal or other co-enzyme
covalently bound to an enzyme
• holoenzyme - a complete, catalytically active
enzyme including all co-factors
• apoenzyme - the protein portion of a holoenzyme
minus the co-factors
• isozyme - (or iso-enzyme) an enzyme that performs
the same or similar function of another enzyme.
This generally arises due to similar but different
genes encoding these enzymes and frequently is
tissue-type specific or dependent on the growth or
developmental status of an organism.
Clinical Use of Enzymes
• Enzyme Activity in Body Fluids Reflects Organ
Status:
• Cells die and release intracellular contents;
increased serum activity of an enzyme can be
correlated with quantity or severity of damaged
tissues (ex. creatine kinase levels following heart
attack)
• Increased enzyme synthesis can be induced and
release in serum correlates with degree of
stimulation (ex. alkaline phosphatase activity as a
liver status marker)
Clinical Use of Enzymes (cont)
• Enzyme Activity Reflects the Presence of
Inhibitors or Activators
• Activity of serum enzymes decreases in presence
of an inhibitor (ex. some insecticides inhibit serum
cholinesterases)
• Determine co-factor deficiencies (like an essential
vitamin) by enzyme activity (ex. add back vitamin
to assay, if activity increases, suggests deficiency
in that vitamin)
Clinical Use of Enzymes (cont)
• Enzyme activity can be altered genetically
• A mutation in an enzyme can alter its substrate
affinity, co-factor binding stability etc. which can be
used as a diagnostic in comparison with normal
enzyme
• Loss of enzyme presence due to genetic mutation as
detected by increased enzyme substrate and/or lack
of product leading to a dysfunction
• NOTE: PCR techniques that identify specific
messenger RNA or DNA sequences are replacing
many traditional enzymatic based markers of genetic
disease
ENZYMATIC REACTION
PRINCIPLES
• Biochemically, enzymes are highly specific for their
substrates and generally catalyze only one type of
reaction at rates thousands and millions times higher
than non-enzymatic reactions. Two main principles
to remember about enzymes are 1) they act as
CATALYSTS (they are not consumed in a reaction
and are regenerated to their starting state) and 2)
they INCREASE THE RATE of a reaction towards
equilibrium (ratio of substrate to product), but they
do not determine the overall equilibrium of a
reaction.
CATALYSTS
• A catalyst is unaltered during the course of a
reaction and functions in both the forward and
reverse directions. In a chemical reaction, a catalyst
increases the rate at which the reaction reaches
equilibrium, though it does not change the
equilibrium ratio. For a reaction to proceed from
starting material to product, the chemical
transformations of bond-making and bond-breaking
require a minimal threshold amount of energy,
termed activation energy. Generally, a catalyst
serves to lower the activation energy of a particular
reaction.
ENZYMATIC REACTION
PRINCIPLES (cont)
• The energy maxima at which the reaction has the
potential for going in either direction is termed the
transition state. In enzyme catalyzed reactions, the
same chemical principles of activation energy and
the free energy changes (DGo) associated with
catalysts can be applied. Recall that an overall
negative DGo indicates a favorable reaction
equilibrium for product formation. As shown in an
enzyme catalyzed reaction, and in the graph, the net
effect of the enzyme is to lower the activation
energy required for product formation.
Chemical Reaction
Enzymatic Reaction Energetics
Reaction Rates
• The rate of the reaction is determined by several factors
including the concentration of substrate, temperature and
pH. For most standard physiological enzymatic reactions,
pH and temperature are in a defined environment (pH 6.97.4, 37oC). Therefore, the concentration of substrate is
the critical determinant. This enzymatic rate relationship
has been described mathematically by combining the
equilibrium constant (the ratio of substrate and product
concentrations), the free energy change and first or secondorder rate theory. The net result for enzymatic reactions is
that the lower the activation energy, the faster the reaction
rate, and vice versa.
Binding Energy
• The graph of activation energy and free energy changes
for an enzymatic reaction also indicates the role binding
energy plays in the overall process. Due to the high
specificity most enzymes have for a particular substrate,
the binding of the substrate to the enzyme through
weak, non-covalent interactions is energetically
favorable and is termed binding energy. The same
forces important in stabilizing protein conformation
(hydrogen bonding and hydrophobic, ionic and van der
Waals interactions) are also involved in the stable
binding of a substrate to an enzyme.
Binding Energy and Transition
State
• The cumulative binding energies from the noncovalent interactions are optimized in the transition
state and is the major source of free energy used by
enzymes to lower activation energies of reactions. A
single weak interaction has been estimated to yield
4-30 kJ/mol energy, thus multiple interactions
(which generally would occur during binding and
catalysis) can yield up to 60-80 kJ/mol free energy this accounts for the large decreases in activation
energies and increases in rate of product formation
observed in enzymatic-catalyzed reactions.
Effect of Temperature
A reaction rate will generally
increase with increasing
Temperature due to increased
kinetic energy in the system until
a maximal velocity is reached.
Above this maximum, the kinetic
energy of the system exceeds the
energy barrier for breaking weak
H-bonds and hydrophobic
interactions, thus leading to
unfolding and denaturation of the
enzyme and a decrease in reaction
rate.
Effect of pH
Variations in pH can affect a
particular enzyme in many ways,
especially if ionizable amino acid
side chains are involved in binding
of the substrate and/or catalysis.
Extremes of pH can also lead to
denaturation of an enzyme if the
ionization state of amino acid(s)
critical to correct folding are
altered. The effects of pH and
temperature will vary for different
enzymes and must be determined
experimentally.
LOCK-ANDKEY
INDUCED FIT
Hexokinase Active Site:
Glucose vs. Galactose Binding
Co-factor:
+
NAD /NADH
(EXAMPLE)
Co-factors: Co-A and Biotin
Catalytic Mechanisms: Types
• Four types of catalytic mechanisms will be
discussed:
• binding energy catalysis
• general acid-base catalysis
• covalent catalysis
• metal ion catalysis
Acid-Base
Catalysis
Many reactions involve the formation of normally unstable, charged
intermediates. These intermediates can be transiently stabilized in an
enzyme active site by interaction of amino acid residues acting as weak
acids (proton donors) or weak bases (proton acceptors). The general
acid and general base forms of the most common and best characterized
amino acids involved in these reactions are shown above.
Acid-Base Catalysis (cont)
• The preceding functional groups can potentially serve
as either proton donors or proton acceptors. This is
dependent on many factors including the molecular
nature of the substrate, any co-factors involved, and the
pH of the active site (which would determine the
ionization state of an amino acid side chain). For acidbase catalysis, histidine is the most versatile amino acid
due to its pKa which means that in most physiological
situations it can act as either a proton donor or proton
acceptor. Generally these amino acids will interact
together with the substrate, or in conjunction with
water or other weak, organic acids and bases found in
cells.
Binding Energy Catalysis
• Binding energy accounts for the overall lowering of
activation energy for a reaction, and it can also be
considered as a catalytic mechanism for a reaction. Several
catalytic factors in the binding of a substrate and enzyme
can be considered: 1) transient limiting of substrate and
enzyme movement by reducing the relative motion (or
entropy) of the two molecules, 2) solvation disruption of the
water shell is thermodynamically favorable, and 3) substrate
and enzyme conformational changes. All three of these
factors individually or in combination are utilized to some
degree by an enzyme. While in some instances these forces
alone can account for catalysis, they are frequently
components of a complex catalytic process involving factors
discussed for the other types of catalytic mechanisms.
Covalent Catalysis
• This mechanism involves the transient
covalent binding of the substrate to an
amino acid residue in the active site.
Generally this is to the hydroxyl group of a
serine, although the side chains of
threonine, cysteine, histidine, arginine and
lysine can also be involved.
Metal Ion Catalysis
• Various metals, all positively charged and
including zinc, iron, magnesium, manganese
and copper, are known to form complexes with
different enzymes or substrates. This metalsubstrate-enzyme complex can aid in the
orientation of the substrate in the active site,
and metals are known to mediate oxidationreduction reactions by reversible changes in
their oxidation states (like Fe3+ to Fe2+).
Summary of Catalytic
Mechanisms
• In general, more than one type of catalytic
mechanism will occur for a particular enzyme
via various combinations of binding energy,
acid-base, covalent and metal catalysis.
Enzymes as a whole are incredibly diverse in
their structures and the types of reactions that
they catalyze, therefore there is also a large
diversity of catalytic mechanisms utilized, the
basis of which must be determined
experimentally.
Substrate Binding Pockets of
Chymotrypsin and Trypsin
Catalytic Mechanism of
Chymotrypsin
Chymotrypsin Mechanism (cont)
Chymotrypsin, last step and
regeneration of active enzyme