Enzymes

Michaelis-Menten Kinetics

• Built on the idea that substrate and enzyme has to first bind and form a complex in order
for the reaction to proceed
• Simple enzyme kinetics involves a single substrate

• rate of reaction is proportional to the substrate and enzyme concentration

Initial velocity-substrate plot

• Looks like a hyperbolic-binding isotherm

• Initially, plot is linear at low substrate concentration
• Michaelis constant (KM) –substrate concentration at which the velocity is half the initial
velocity
• Maximum velocity – maximum initial velocity attainable at very high substrate
concentration

Michaelis-Menten mechanism
 • recognition of the substrate is separated from the catalysis of the chemical reaction.
• Rate-determining step is the second reaction

• By steady-state approximation

• Maximum velocity is dependent on the rate constant of the second reaction and total
enzyme concentration

• Hyperbolic initial velocity vs. substrate concentration plot is not recommended because
velocity must also be observed at high substrate concentrations to see the behavior
o Not possible if substrate is not sufficiently soluble
• Michaelis constant can be interpreted as a dissociation constant

o The higher it is, the less affinity the enzyme has for the substrate
o If k2 is very small, Michaelis constant is approximately the equilibrium constant of
the dissociation of ES to E + S. Product formation is very slow
o If k2 is very large, rate-determining step is fast and complex is quickly converted
to free enzyme and products. Occupancy of active site decreases
• At low S, velocity has linear dependence
• At high S, most enzymes are occupied, so increase in rate decreases.

Turnover number and Catalytic Efficiency

• At low substrate concentrations, rate is pseudo-first order

o k2/kM is a rate constant for pseudo-second order

• At high substrate concentrations, rate is pseudo-zero order, equal to maximum velocity:

• k2 = Vmax/[E]0 = turnover number or catalytic rate constant (kcat)

o maximum number of reactions per mole of enzyme
 o kcat = k2 for simple Michaelis-Menten kinetics, but for complex mechanisms it can
be related to more than one rate constants
• k2/kM or kcat/kM is catalytic efficiency
o ratio of the turnover number to the Michaelis constant
o at low substrate concentrations, it is equivalent to a second-order rate constant
o Increase in catalytic efficiency increases efficiency of the reaction
o High catalytic efficiency means catalytic step (2nd step) is very rapid

The perfect enzyme

• Ideal enzyme is one that catalyzes the chemical step of the reaction as fast as the
substrate can get to the enzyme
• kcat tells how fast enzyme catalyzes the reaction once substrate has arrived at the active
site.
• Enzymes need to be very fast to keep up with demands of the cell (e.g. ATP production,
fight-or-flight response)
• rate of reaction should be diffusion-controlled
o rate depends on the collision of substrate with active site if diffusion-controlled
o Some enzymes can work faster than the diffusion limit by electrostatic focusing
o Electrostatic focusing uses highly polarized groups to guide positively charged
substrate faster to the active site
o Electrostatic steering draws substrate that has a same charge as the enzyme to
the active site of opposite charge

Enzyme specificity

• Specificity depends on rate of catalytical step and Michaelis constant

• There will be different catalytic efficiencies for different substrates
o this is why catalytic efficiency is also a specificity constant!
o highest value of catalytic efficiency will be degraded most rapidly

Graphical analysis

• Lineweaver-Burk (double-reciprocal plot)

 o Advantage: no need to measure rates at very high substrate concentrations to get
value of maximum velocity
o Overemphasizes data at low substrate concentration

• Eadie-Hofstee plot (v0 vs. v0/S)

o Gives equal weight to data points in any range of substrate concentration and
reaction rate. However there is overemphasis to smaller values of v0/S
o Disadvantage is that both are dependent on reaction rate. Experimental error will
be present in both axes.

• Hanes-Woolf plot (S/v0 vs. 1/v0)

 odoes not overemphasize the data at low substrate concentration
oDisadvantage is that both are dependent on substrate. Experimental error will be
present in both axes.
• These plots assume Michaelis-Menten kinetics. More complex mechanisms for oligomeric
enzymes do not apply.

Inhibition

• Competitive – Inhibitor binds to enzyme in on near active site

o Substrate cannot bind since inhibitor blocks it

o Competition of active site between inhibitor and substrate
o Less free enzyme available for substrate binding, thus rate of product formation
decreases
o Inhibitor does not undergo chemical reaction due to chemical structure
differences to the substrate
o Inhibitor may or may note have similar structure to that of the substrate
o Maximum rate is not affected (can be achieved if there is no inhibitor at high
substrate concentration)
o Michaelis constant increases since there is less free enzyme available, and higher
substrate is needed to raise the velocity to half of its maximum value
 o Lineweaver-Burk plot shows that inhibition shifts the line counterclockwise, with
the y-intercept as the pivot point
o at low concentrations or if dissociation constant of inhibitor-enzyme complex is
large, then kinetics is approximately simple Michaelis-Menten kinetics
o if there is inhibition, Michaelis-Menten behavior still applies but at higher
Michaelis constant
o At very large inhibitor concentrations, most inhibitors will bind to enzyme leaving
no room for substrate
• Uncompetitive inhibition
o Inhibitors bind to the enzyme away from the active site
o Presence of substrate does not affect binding of inhibitor
o However, inhibitor binds to the ES complex, not allowing the substrate to be
converted.

o Michaelis constant is decreases because inhibitor prevents conversion of

substrate. This makes the enzyme just bind to substrate unreacted, increasing its
affinity.
 o Maximum velocity is expected to decrease since inhibitor sabotages the ES
complex, forming less products

o Catalytic efficiency is unaffected!!!

o Lineweaver-Burk plot shows that inhibition shifts the line to the left but parallel
to uninhibited line
• Noncompetitive inhibition
o Also known as mixed inhibition
o Inhibitor can bind at enzyme or at enzyme-substrate complex

o Maximum velocity is expected to decrease

o However, Michaelis constant can either increase, decrease, or remain unchanged!
o Increase in Michaelis constant – if inhibitor has greater affinity to enzyme. Line in
Lineweaver-Burk plot shifts counterclockwise with a pivot point somewhere in 2nd
quadrant
 o Decrease in Michaelis constant – if inhibitor has greater affinity to complex. Line
in Lineweaver-Burk plot shifts line to the left but not parallel to uninhibited line
o No change – if inhibitor has equal affinity to either enzyme or complex (Pure
competitive inhibition). Line in Lineweaver-Burk plot shifts counterclockwise with
a pivot point at the x-intercept.

Inhibitor classes

• Reversible – allows binding of the substrate to form products

• Irreversible – block substrate binding permanently by reacting covalently at an active site
• Suicide substrates – inhibitors that trigger catalytic cycle but leaves a dead-end complex

Ping-Pong mechanism

• Limitations of Michaelis-Menten kinetics is that there is only one substrate

• Many biological processes involve multiple substrates

• First substrate reacts to enzyme to form product

• Enzyme is structurally modified for second substrate to react
• Enzyme reverts to its original form to react with the first, and process repeats
• Lineweaver-Burk plot should show vertical set of parallel lines for different first substrate
concentrations at constant concentration of the other

Random mechanism

• Either one of the substrates can bind first and one of the products can be released first
 • binding of one substrate does not significantly affect the binding of the other

Ordered mechanism

• There is a specific order for substrate binding and product release

• Order is determined by changes in conformation and affinity of the enzyme to the

substrate
• First substrate must enhance the affinity of the second substrate to the enzyme
• Common in allosteric enzymes
• Michaelis constant will be dependent on both substrates

Allosteric enzymes

• Enzymes with multiple binding sites can exhibit allostery

• Substrate binding to an enzyme can be cooperative, inducing conformational change to
the other subunits of the enzyme
• Substrates, products, or other molecules unrelated to the two can allosterically affect the
enzyme
• Velocity-concentration plot becomes sigmoidal instead of hyperbolic

Transition state stabilization

• rate constant depends on activation energy by Arrhenius’ Law

• ES complex to EP complex is usually a rate determining step
o transition state has very high activation energy
• Optimum interaction occurs when active site is complementary to transition state
o geometrical match of TS to active site
o charges for electrostatic interactions
o optimized hydrogen bonds between TS and active site
o complementarity lowers transitions state’s activation energy
• Electrostatic effects are very important
o due to many reactions that cause charge distribution changes in enzyme or
substrate
o due to longer range and larger in magnitude
• TS stabilization is not always the most important factor, though it is often the most
dominant contributor

Enzymes as acids or bases

 • It is difficult for cells to change the pH of the whole cytoplasm
• AA residues in enzymes can act as acids or bases depending on their side chains and if
they are terminal residues:
o Acidic: Asp, Glu
o Basic: His, Lys
o Polar Neutral: Cys, Tyr (rare)
o N-terminus or C-terminus
• Substrate can shift the pKa value of the AA side chain or terminus
o original protonation state goes back again after substrate binding ends
• There is an optimum pH for enzymatic reactions (e.g lysozymes)
o there should be a balance of protonated and unprotonated residues

Proximity effects

• Involves entropy
• Substrate molecules react or organize a substrate to a specific conformation optimal for
intermolecular reaction.
• Enzymes use binding free energy to compensate for the lost of translational of rotational
freedom upon binding with substrate
• control of relative orientation may be important for controlling the stereochemical
outcome

Chemical Rxns in Enzymes

• nucleophilic substitutions
• acid-base catalysis
• metal-ion catalysis

Absolute and Relative Specificity

• absolute – catalyzes the reaction of one, and only one substrate to a particular product
has a fairly rigid active site
o lock-and-key model
• relative – can catalyze substrates that are structurally related to each other
o active sites are more flexible
o induced fit model
• stereospecific enzymes – has asymmetric binding site to accommodate chiral substrates
o binding site must not have the shape of the substrate’s mirror image
o gives a product with a specific stereoisomerism

Control of Enzyme Activity by Phosphorylation

• Phosphorylation – side chain OH groups of Ser, Thr, and Tyr can form phosphate esters
 o important in Na-K pump
o source of phosphate group is ATP
o ATP hydrolysis to ADP releases energy to couple nonspontaneous biological
reactions
o ATP donates phosphate to aspartate, causing conformational change in enzyme
• Protein kinases – catalyze phosphate donation to enzymes
• Phosphorylation may or may not increase enzyme activity
• Phosphatase – catalyze release of phosphate bonded to enzyme

Serine proteases

• contains conserved Ser-His-Asp catalytic triad

o conserved on all serine proteases!!!
• cleaves peptide bonds by adding water across the bond
o slow due to large activation energy and low concentration of -OH attacking group
o nonspecific cleavage in water
• serine proteases lower activation energy
• serine proteases provide high concentration of attacking nucleophile
• serine proteases provide specificity in peptide cleavage
• ping-pong type mechanism

Chymotrypsin action

• serine’s -OH is hydrogen-bonded to histidine’s nitrogen

• tetrahedral intermediate forms by nucleophilic substitution to the carbonyl group of
substrate while serine is deprotonated and histidine is protonated (acid-base)
• C-N bond is cleaved and histidine is deprotonated, forming acyl-enzyme complex and the
cleaved residue
• Water reacts via nucleophilic substitution while histidine gets proton from water
• tetrahedral intermediate forms
• serine is protonated and histidine is deprotonated while C-O bond of substrate to
enzyme is cleaved, forming the second residue
Specificity Pocket

• specificity “pahket”
• sidechain of residue before the would-be cleaved amide bond fits into pocket
• relatively long, narrow, and hydrophobic except an aspartate residue for trypsin
o aspartate acts as salt bridge for the intermediate
o pocket can have unique residues, depending on cleavage pattern
• specificity pocket can only accommodate certain AA residues
o it determines the cleavage pattern of an enzyme
o ex: trypsin’s specificity pocket only accommodates Lys/Arg

Oxyanion hole

• lowers energy of the transition state

• allows negatively-charged oxygen to form hydrogen bonds with -NH groups, stabilizing
the transition state

Zymogens

• Inactive precursor of an enzyme

• Irreversible transformation to an active enzyme by cleavage of covalent bonds
• Proteases can cleave zymogens to make them active
• Trypsinogen and chymotrypsinogen
o zymogens of trypsin and chymotrypsin
 o Found in pancreas (they can’t be active inside pancreas!)
o Trypsin catalyzes activation of chymotrypsinogen
o Enteropeptidase catalyzes activation of trypsinogen
o π-chymotrypsin then reacts in itself to form α-chymotrypsin, the final form
• activation of zymogens changes the tertiary structure

Coenzymes

• Cofactors - nonprotein substances that take part in enzymatic reactions and are
regenerated for further reaction
o examples: metal ions in carboxypeptidase
o metal-ion coordination allows specific geometries that aid positioning of groups
for optimum catalysis
• Coenzymes – nonprotein organic cofactors
o examples: vitamins and their derivative
o most are involved in redox reactions and group transfer in metabolic processes

• NAD+
o nicotinamide adenine dinucleotide
o redox coenzyme
o composte of adenine, ribose, two phosphate groups bonded together, another
ribose and nicotinamide
o redox site occurs at nicotinamide ring

• Vitamin B6
o pyridoxal, pyridoxamine, pyridoxine and their phosphorylated forms
o group-transfer coenzyme
 o transfer amino groups from one molecule to another for AA biosynthesis (group
donor and group acceptor)

Ribozymes

• catalytic RNAs
• RNAs in cell are bound to proteins in ribonucleoprotein complexes
o catalyze protein synthesis and mRNA splicing
o other processes include RNA processing, protein translocation, gene silencing,
RNA export, and addition of telomeric DNA
• most catalyze phosphoryl transfer reactions
o require activation of either the 2’-hydroxyl group in ribose or a water molecule for
nucleophilic attack of a phosphodiester bond