Enzymes:

•Enzymes are biological catalysts.

•They increase the rate of a biochemical reaction and are regenerated to their original form at the end of
the process.
•They do not affect the equilibrium state or the thermodynamics.
•Enzymes are proteins, and their function depends upon their structure.
Enzyme Nomenclature
•Enzyme names often end in “–ase.”
•“-ase” generally follows the type of reaction or the name of the substance involved.
•There is a systematic name for every enzyme that specifically identifies it.
•This IUBMB naming system is precise but is very cumbersome.
Substrate
•“Substrate” refers to the principle substance or substances involved in the reaction (the reactants).
•There are often other molecules (e.g. water) that are involved in the reaction as well.
•Substrates bind to the active site of the enzyme molecule.
Coenzymes
•Coenzymes are small, non-protein molecules that act with specific enzymes as participants in the
reaction.
•They are changed in the reaction.
•Another reaction regenerates them to their original form.
•Vitamins often make up part of their structure.
Prosthetic Group
•A prosthetic group is a small molecule that participates in the reaction and is tightly bound to the
enzyme.
•It is also changed in the reaction.
•The enzyme catalyzes another reaction that regenerates the prosthetic group to its original form.
Holoenzyme and Apoenzyme
•“Holoenzyme” refers to the enzyme protein and its associated prosthetic group.
•“Apoenzyme” refers to just the enzyme protein without the prosthetic group.
Classes of Enzymes
•Oxidoreductase- Catalyzes oxidation reduction reactions.
•Transferase- Catalyzes the transfer of a chemical group containing C, N, or P to another molecule.
•Hydrolase- Catalyzes the cleavage of a bond using a water molecule.
•Lyase- Catalyzes the cleavage of C-C, C-S and some C-N bonds.
•Isomerase- Catalyzes the conversion of a molecule to one of its isomeric forms.
•Ligase- Catalyzes the formation of C-O, C-S and C-N bonds.
EnzymeActive Site
•This is a place on the enzyme molecule where the reaction occurs.
•The substrates (reactants) bind to this site and form a transient Enzyme Substrate Complex [ES].
•The [ES] is a transient state during which the actual reaction occurs.
•The enzyme then releases the product.
Formation of ES Complex

Enzyme Specificity
•Enzymes usually catalyze only one chemical reaction.
•They often work with only one substrate or a very limited number of substrates.
•For example, only L isomers of amino acids react with the enzyme that catalyzes protein synthesis.
Cofactors
•These are molecules necessary for proper enzyme activity.
•They may participate in the reaction (i.e. coenzymes).
•For example, metallic ions may be necessary to maintain proper enzyme structure.
•They may be involved in binding the substrate to the active site.

Chemical Reactions
•A chemical reaction involves breaking of existing chemical bonds and forming new ones.
•In order to react, the molecules must collide with sufficient energy to break the old bond.
•The collision results in the formation of a new substance called the transition state.
•The reaction proceeds from the transition state.
•The minimum amount of energy required to form the transition state is called the activation energy.
•The molecules must collide in the proper orientation so the correct bond is broken.
Enzyme Action
•The enzyme specifically binds the substrate molecules to the active site in the proper orientation.
•The enzyme-catalyzed reaction requires less collision force than the non-catalyzed reaction.
•The enzyme lowers the activation energy requirement for the reaction.
Activation Energy Required

Equilibrium State
•Biochemical reactions are reversible.
•When the reaction is allowed to proceed, an equilibrium state is reached.
•In the equilibrium state, the forward reaction and the reverse reaction occur at the same rate.
•No more net product is formed.
Equilibrium Constant
•At equilibrium there is a fixed ratio of product to reactants.
•Enzymes do not change this ratio.
•Enzymes increase the rate at which the equilibrium state is reached.

Keq = [P] / [S]

Monkeys and Peanuts: A Model
• Assume we have a troop of ten monkeys.
• All are equally adept at finding and shelling peanuts.
• The monkeys are too full to eat peanuts but are compelled to go on shelling.
• The peanuts are a new hybrid variety that can just as readily be stuck back together again.
Initial Conditions
• Peanuts are spread uniformly around the assay room, but the number varies in different experiments.
• The “concentration” of peanuts depends upon the number of peanuts and the total floor space.
• Initially, the monkeys are kept in an adjacent room.
• The assay begins when the monkeys are allowed into the assay room.
Assay One
• The initial peanut concentration is 1 peanut per square foot.
• The average monkey spends 9 seconds finding a peanut and 1 second shelling it.
• The average shelling rate is 0.1 peanuts per second per monkey.
• The total rate is 1.0 peanuts/sec.
Assay Two
• The peanut concentration is 3 peanuts per square foot.
• The average monkey spends 3 seconds finding a peanut and 1 second shelling it.
• The average shelling rate is 0.25 peanuts per second per monkey.
• The total rate is 2.5 peanuts/sec.
Assay Three
• The peanut concentration is 9 peanuts per square foot.
• The average monkey spends 1 second finding a peanut and 1 second shelling it.
• The average shelling rate is 0.50 peanuts per second per monkey.
• The total rate is 5 peanuts/sec.
Ultimate Assay
• At some peanut concentration, the monkey takes no time to find a peanut.
• The peanuts are so numerous that every time the monkey drops a shelled peanut, another appears in its
hand.
Maximum Velocity
• The time to find a peanut is 0 sec, and the time to shell it is 1 sec.
• The average rate is 1.0 peanut per sec per monkey.
• The total rate (velocity) is 10 peanuts per second.
• For 10 monkeys, this is the maximum velocity
• Further increases in concentration of peanuts will not result in increased velocity.
Enzyme Rate Curve

Enzyme Catalyzed Reactions

Rate Constants

Substrate Depletion Equals Product Formation

V = -d[S]/dt = +d[P]/dt
Michaelis-Menton equation

Very Low Substrate Concentration

V = Vmax [S]/ Km+[S] = Vmax [S]/ Km; V= Vmax [S]/ Km

Very High Substrate Concentration

V = Vmax [S]/ Km+[S] = Vmax [S]/ [S] = Vmax; V=Vmax

Substrate Concentration Equals Km

V = Vmax [S]/ Km+[S] = Vmax [S]/ 2[S] = Vmax/2; V=Vmax/2

Lineweaver Burke Equation

• It converts the hyperbolic relationship of the Michaelis – Menton equation into a linear relationship by
taking the reciprocal of both sides of the equation.

Double Reciprocal Plot

• The graph is plotted as 1/v versus 1/S.
• The y intercept is 1/Vmax.
• The x intercept is -1/Km

Enzyme Kinetics
• Turnover Number- Kcat- This is the number of substrate molecules converted to product in a unit of time
for each enzyme molecule.
• Specificity Constant- This is the ratio between Kcat and Km
Enzyme Catalysis
Covalent Interactions
• Rearrangement of covalent bonds between substrate and enzyme amino acid functional groups occurs.
• Formation of transient covalent bonds produces a reaction pathway with a lower activation energy
requirement.
Non-covalent Interactions
•Formation of an ES complex involves weak interactions (e.g. hydrogen bonds, hydrophobic interactions,
and ionic bonds).
•The ES complex is a stable intermediate with a slight decrease in free energy.
•This decrease is called the binding energy.
Binding Energy
•The binding energy contributes to the catalytic power of the enzyme.
•It also induces conformational changes in the enzyme structure relative to the substrate shape.
•Weak interactions are optimized in the reaction transition state.

Formation of Transition State

•Lock and Key Theory
•Induced Fit Theory
Non-Catalyzed Reaction

Lock and Key Theory

•In this theory, there is strict complementarity between the shape of the enzyme’s active site and the
substrate.
•It does not provide a reasonable mechanism for catalysis.
•It is an old idea that is now discounted.
Induced Fit
•In this theory, there is loose complementarity between the enzyme and the substrate.
•Once the ES complex forms, a weak interaction causes a conformational change.
•This change brings the ES complex to the transitional state.
•Energy is released in forming the transition state, which lowers the activation energy requirement of the
reaction.

Enzyme Specificity
• Enzymes can discriminate between different potential substrates.
• An enzyme active site has functional groups that bond with functional groups of the substrate molecule.
• Specificity is derived from the formation of multiple weak interactions between the enzyme and the
substrate.

Mechanisms of Catalysis
•Acid – Base Catalysis
•Covalent Catalysis
•Metal Ion Catalysis
General Acid- Base Catalysis
•In this mechanism there is formation of an unstable, charged intermediate that breaks down into the
original reactants.
•Water can be a proton donor or acceptor to stabilize the charged intermediate.
•This allows the reaction to proceed.
Enzymatic Acid- Base Catalysis
•The active site of the enzyme can contain amino acid side chains that can be either proton donors or
proton acceptors.
•They can be precisely located to allow proton transfer expeditiously.
Covalent Catalysis
•A transient covalent bond is formed between the enzyme and the substrate.
•An example is amino acyl tRna synthetase.
•A new pathway with a lower activation energy requirement is present.
•The transient complex undergoes further reaction to regenerate the enzyme.
Metal- Ion Catalysis
•Many enzymes require metal ions in order to function.
•The metal can help bind the substrate to active site of enzyme, contributing to the binding energy.
•The metal can directly participate in oxidation – reduction reactions.
•Fe+++ + e- → Fe++
Enzyme Catalysis

Effect of Temperature
•The rate of the reaction increases as temperature increases, until a maximum velocity is reached.
•The temperature at which the maximal velocity is reached is termed optimal temperature.
•Above optimal temperature, the rate decreases.
•The initial increase in the rate is due to increased kinetic energy of the reactants.
•The subsequent decrease in the rate above optimal temperature is due to thermal denaturation of the
enzyme.

Effect of pH
•The maximum reaction velocity occurs at a specific pH.
•The availability of protons determines the charge in functional groups of various amino acids that make
up the active site of the enzyme.
•Extremes of pH can denature the enzyme.
Enzyme Regulation Mechanisms
Chymotrypsin
• Chymotrypsin is a pancreatic protein digestive enzyme.
• It catalyzes the hydrolysis of the peptide bond adjacent to an aromatic amino acid.
• It enhances the reaction rate by a factor of 109.
• The reaction occurs in two steps: Acylation, Deacylation
Free Enzyme
• The hydroxyl group of Ser195 is hydrogen bonded to His57.
• His57 is also hydrogen bonded to Asp102.
• The aromatic ring of the substrate fits into a hydrophobic pocket on enzyme.
• The carbonyl oxygen of the peptide bond to be broken fits into an oxyanion hole.

Acylation
• The oxygen of Ser195 performs a nucleophilic attack on the carbonyl carbon atom.
• His57 acts as a base to accept a proton from Ser195.
• During the nucleophilic attack, the carbonyl oxygen acquires a negative charge.
• This intermediate is stabilized by hydrogen bonds from amides of the enzyme’s peptide bonds.
Deacylation
• This process is the reverse of the first step.
• Water provides the proton in place of the amide of the peptide bond that was broken.

Regeneration of Enzyme

Hexokinase

•This reaction catalyzes the transfer of phosphate groups from ATP to glucose in intermediary
metabolism.
• It uses atoms of Mg++ to stabilize a charged transition state.
• Without the substrate, the enzyme assumes an inactive conformation.
• The binding of ATP, glucose, and Mg++ causes a conformational change in the enzyme to its catalytically
active shape.
Enolase

•This enzyme catalyzes a reaction of glycolysis.

• Lys345 acts as a base catalyst in abstracting a proton from C2 of 2-phosphoglycerate.
• Strong interactions from the two Mg++ ions make the C2 carbon give up its proton more readily.
• Glu211 acts as a general acid catalyst, donating a proton to the leaving OH group.

Lys345 acts as a Base catalyst Glu211 acts as an Acid catalyst

Product of Reaction
Enzyme Regulation
Classification
• Inhibition of Enzyme Activity
– Competitive inhibition
– Noncompetitive inhibition
– Mixed inhibition
• Regulation of Enzyme Activity
– Allosteric binding sites
– Regulation of enzymes by reversible covalent modification
– Induction and repression of enzyme synthesis
Competitive Inhibition
• This occurs when the inhibitor binds reversibly to the same site that the substrate would normally
occupy.
• At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the
absence of inhibitor.
• A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the
presence of a competitive inhibitor, more substrate is needed to achieve ½Vmax.

Statin Drugs are Competitive Inhibitors

• This reaction is catalyzed by hydroxymethylglutaryl–CoA reductase (HMGCoA reductase).
• Statin drugs, such as atorvastatin (Lipitor) and simvastatin (Zocor), are structural analogs of the natural
substrate for this enzyme.
• They compete effectively to inhibit HMG-CoA reductase.
• By doing so, they inhibit cholesterol synthesis, thereby lowering plasma cholesterol levels.
Noncompetitive Inhibition
• Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme.
• The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the
reaction from occurring.
• These inhibitors decrease the apparent Vmax of the reaction.
• They do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in
the presence or absence of the noncompetitive inhibitor.

Mixed Inhibitors
• These affect both the Km and the Vmax.
• They affect the binding of the substrate and the rate at which the enzyme can convert substrate to
product.

Enzyme Inhibitors as Drugs

• At least half of the ten most commonly dispensed drugs in the United States act as enzyme inhibitors.
• For example, the widely prescribed β-lactam antibiotics, such as penicillin and amoxicillin, act by
inhibiting enzymes involved in bacterial cell wall synthesis.
Regulation of Enzyme Activity
• The rates of most enzymes are responsive to changes in substrate concentration.
• Some enzymes with specialized regulatory functions respond to allosteric effectors or covalent
modification.
• Others show altered rates of enzyme synthesis (or degradation) when physiologic conditions are
changed.
Allosteric Enzymes
• The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or modify the
maximal catalytic activity of the enzyme, or both.
• Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme
activity are called positive effectors.

• When the substrate itself serves as an effector, the effect is said to be homotropic.
• The effector may be different from the substrate, in which case the effect is said to be heterotropic.

Regulation of Enzymes by Reversible Covalent Modification

• This most frequently occurs by the addition or removal of phosphate groups from specific serine,
threonine, or tyrosine residues of the enzyme.
• Protein phosphorylation is recognized as one of the primary ways in which cellular processes are
regulated.

• Depending on the specific enzyme, the phosphorylated form may be more or less active than the
unphosphorylated enzyme.
• Phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity.
• The addition of phosphate to glycogen synthase (an enzyme that synthesizes glycogen) decreases
activity.
Induction and Repression of Enzyme Synthesis
• The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total
population of active sites.
• For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the
synthesis of key enzymes involved in glucose metabolism.
Ethylene Glycol (anti-freeze) Ingestion
A 6-year-old boy is brought to the ED because of slurred speech, lethargy, and severe vomiting. He drank
some sweet liquid while helping his father in the garage. Shortly after arrival he had a seizure. His blood
pressure is low and he is breathing rapidly. His urine sediment shows oxalate crystals.
Discussion: Toxicity of ethylene glycol and methanol is due to their conversion by alcohol dehydrogenase
to toxic substances. Treatment includes administration of a substance that has a higher affinity (lower
Km) for the alcohol dehydrogenase, which allows the ethylene glycol or methanol to be harmlessly
eliminated in the urine. Traditionally, ethanol has been used to block alcohol dehydrogenase, but
fomepizole (trade name Antizole) is more commonly used now.
Ethanol Intoxication
A 22-year-old college student who typically binge drinks at the end of each semester presents with a blood
alcohol level of 200mg/dl (0.2%). How many hours must pass before he is under the legal limit for driving?
Discussion: Ethanol is metabolized initially by first-order kinetics. As the relevant enzyme becomes
saturated it moves to zero-order kinetics (fixed amount metabolized per unit time). At that point, the
level will fall by 15-45 mg/dl/hour depending on the chronicity of ethanol use. The emergency physician
can reasonably assume the rate of metabolism will be in the higher range (25-45mg/dl/hr) for known
habituated drinkers.
Using the above information, one can estimate how long it will take a patient to become legal for driving
based on the severity of his or her alcoholism and the blood alcohol level. Our 22-year-old college student
will most likely be under the legal limit for driving (80mg/dl, or 0.08%, in most states) in about six hours.
To be on the safe side, you should repeat a blood alcohol level before discharge if the patient is going to
be driving.
Methemaglobinemia
A newborn male in the nursery is noted to be cyanotic (blue), though he doesn’t appear to be in any
distress. That morning, benzocaine had been used as a local anesthetic during his circumcision. His pulse
oximetry value is 85%, and his chest x-ray is normal. His arterial blood looks like chocolate syrup when
it’s drawn into tubes.
Discussion: Methemoglobin is an oxidized Fe+3 (ferric) form of hemoglobin that is unable to carry O2 or
CO2 effectively. Normal is Fe+2 (ferrous). Benzocaine is an oxidizing drug that can lead to
methemoglobinemia. Catalysis is changed by the oxidation state of the metal ion in this case. A way to
support the diagnosis is to place 1-2 drops of blood on white filter paper, and then evaluate for color
change upon exposure to oxygen (this test can be accelerated by gently blowing supplemental oxygen
onto the filter paper). Deoxygenated hemoglobin changes from dark red or violet to bright red, whereas
methemoglobin remains brown.
Temperature affects rate of catalysis: Hypothermia
Hypothermia progressively depresses the CNS (Central Nervous System), decreasing CNS metabolism in a
linear fashion as the core temperature drops. At core temperatures less than 33°C, brain electrical activity
becomes abnormal; between 19°C and 20°C, an electroencephalogram (EEG) may appear consistent with
brain death. . . . A patient's companions often note initial symptoms in the field. Symptoms can include
mood change, irritability, poor judgment, and lassitude. Companions may note rhythmic or repeated
motions such as rocking. Slurred speech and ataxiamay mimic a stroke, alcohol intoxication, or high-
altitude cerebral edema. It’s often their poor judgment that kills hypothermic people.
Aspirin irreversible Platelets last ten days—ten percent of platelets replaced each day. It is desirable to
stop aspirin several days before surgery to allow bleeding parameters to normalize.
Ibuprofen reversible Because the platelets recover when the medication is cleared, bleeding parameters
normalize within 12 to 24 hours.