I. ENZYMES: PROTEINS THAT CATALYZE REACTIONS
Catalytic Power- ability to increase reaction rates
A. Most reactions in biological systems occur slowly in the absence of catalysts; for practical
purposes the rate is zero. Reactions are not at equilibrium except as enzymes move them toward EQ.
B. Fastest known enzyme: carbonic anhydrase: each molecule catalyzes 600,000 times per second
(=107 X as fast as uncatalyzed). (Table 14-1, page 480 kcat)
II. Specificity toward substrates (the reactants)
A given enzyme will catalyze a single reaction (or, in some cases, a single type of reaction). To be so
specific, it must "recognize" its substrates and not interact strongly with the other molecules it
encounters. The specificity is determined by complimentary 3-D shape (or “fit”: square peg can't fit
round hole) AND reversible interactions. (fig 13-1, p460) Examples:
1. There are a variety of proteolytic enzymes, which catalyze hydrolysis of peptide bonds of other
proteins (as in digestion of food). Some of these will cleave bonds only on a particular side of a
particular type of AA residue, others between two particular residues.
2. Most enzymes that act on AAs are specific for the L isomer. Beyond this, they are
stereospecific in that they will specifically remove one of 2 "equivalent" groups from a symmetric
molecule.
III. Regulation-Control of the activity of enzyme.
If all molecules of a given enzyme are inactivated, reaction rate approaches zero. If all are activated
(or not deactivated), reaction proceeds toward EQ at high rate. We shall see that usually control of
one reaction in a pathway results in control of all of them.
Types:
1. Allosteric:
a. as with Hb: small indicator molecules
b. Regulatory proteins are themselves regulated and then act to control other proteins.
2. Covalent modification: often a hydroxyl containing residue (ser, thr, tyr) will be phosphorylated at
its –OH group. This results in stabilization of one form of the protein, as in CO2 effect on Hb.
3. Proteolytic activation: the enzyme is synthesized in inactive form, becomes active after a
segment of its polypeptide chain is removed. (common for food digesting proteases)
IV. Enzymes interconvert forms of energy. Living things are "chemical engines". Foods are energy
fuels; the energy released when they are metabolized is used as mechanical (muscle contraction),
osmotic (nutrient transport), chemical (biosynthesis), etc. energy.
V. Energy-Thermodynamics: (skip First Law)
A. Second Law: For all spontaneous processes, the entropy of the universe increases. In
biosynthetic processes the entropy decreases, but this is offset by a larger increase in the entropy of
surroundings. (ΔS (universe) = ΔS (system) + ΔS (surroundings) )
B. The above statement of the Second Law is difficult to use practically:
1. It can be converted mathematically to the following result: a reaction occurs spontaneously
only if ΔG < 0. It is at equilibrium if ΔG = 0. (ΔG is the "free energy change") (spontaneously means left
to right. We say the reaction is not spontaneous if ΔG > 0, but what this means is that the reaction
proceeds from right to left as written.)
2. We will often be concerned with readily reversible reactions. The reaction proceeds at a
significant rate from right to left and from left to right: at equilibrium the rates of the reactions in each
direction are equal and ΔG = 0. If ΔG < 0 the rate of the reaction to the right is greater than that to
the left and we say the (net) “reaction goes to the right”” (until EQ is reached). If ΔG > 0 we say the
(net) “reaction goes to the left”. (Note: Spontaneously means "without proportional input of energy". It
does not mean "occurs immediately" or "occurs rapidly". There is no relationship between ΔG and
rate.)
3. ΔG is independent of path. The ΔG for glucose oxidation is the same whether the glucose is
burned in air (a reaction in which oxygen molecules collide with glucose molecules) or occurs in a
living system without flame or any direct contact between oxygen and glucose.
C. For the reaction: aA + bB ---> cC + dD; ΔG = ΔGo' + 2.303 RT log Q; where
Q = [C]c[D]d/[A]a[B]b at the existing conditions and ΔGo' = -2.303 RT log Keq. The superscripts on ΔG o'
indicate standard conditions, namely [A] = [B] = [C] = [D] = 1 M (if A, B, C, and/or D are aqueous
substances) (and if A, B, C, or D is a gas, PA =PB = PC = PD = 1 atm for a gas), and the prime indicates
a special standard condition for biochemistry: pH = 7. The value of ΔGo' indicates the spontaneous
direction of the reaction under standard conditions as for ΔG above.
Note that to calculate ΔGo' we must first calculate Keq from the EQ concentrations of A, B, C, and D.
If the system is at EQ: ΔG = 0 and Q = Keq. (Note: R= 8.31 J/molK = 8.31X 10-3 kJ/molK and 2.303RT
= 5.7 kJ at 298K.)
1. Definition: "position of equilibrium" is the relative amounts of reactants and products at EQ.
a. If, at EQ, the concentrations of C and D are greater than those of A and B (actually [C][D] >
[A][B]) then "products are favored", Keq>1 and ΔG o' <0.
b. If K<1, log K<0, ΔGo' >0, and "reactants are favored" ([C][D] < [A][B]).
c. ΔG = -5.7 kJ if Keq = 10 and log Keq=1. For each 5.7kJ change in ΔG o', the Keq changes
by a factor of 10. see table 3-3, p49
2. Note that the spontaneity of a reaction depends on the 2 factors in the ΔG expression:
i.
ΔGo' indicates the characteristics of the substances in the reaction regarding which are more stable
and favored at equilibrium.
ii.Q indicates existing concentrations.
If Q=10 and Keq=3 then there are more products (and/or less reactants) than at EQ and the reaction
will go left even though products are favored.
VI. Kinetics: effects of enzymes on reaction rates.
A. 1. Enzymes have no effect on ΔGo', ΔG, Keq, or the position of EQ.
2. Enzymes cannot make a reaction go in a certain direction, they only make it go faster in the
direction (net) specified by ΔG.
3. Enzymes actually accelerate the rate in both directions equally (by an equal factor).
4. They increase the rate at which the reaction goes toward EQ. Most biological reactions are
not at EQ in a living cell.
B. Enzymes accelerate the reaction by lowering the activation energy of the reaction. They do this
by stabilizing the transition state.
1. What does this mean? And how do enzymes do it? By forming bonds with the transition
state that they don't have with reactants or products.
a. Recall that bond formation releases energy, leaving the bonded substances at a lower
(more stable) energy.
b. Example: tyrosyl-amino acyl tRNA synthetase.
i. In this reaction, the carboxyl O of tyrosine is to become linked to the first phosphate of
ATP (adenosine triphosphate) with the other two phosphates being released.
ii. The tyr and nonphosphate portion of ATP are bound throughout the reaction. But the
third phosphate of ATP is bonded only in the transition state.
iii. The geometry at P of the first phosphate is tetravalent (tetrahedral) in reactants and
products, but it's pentavalent (trigonal bipyramidal) in the transition state.
iiii. When the geometry is pentavalent, the end phosphate is placed where it can H-bond to
two of the enzymes side chains at the active site. This is the only time these bonds are made.
2. This is called specific binding of the transition state.
VII. Molecular Mechanism of Allosteric Regulation of Enzyme Activity
(Covalent modification by phosphorylation of side chain OH groups results in similar effects)
A. Example Enzyme (E): Aspartate Transcarbamoylase (ATCase)
1. Catalyses the first reaction in pyrimidine nucleotide synthesis, linking the substrates aspartate
(asp) and carbamoyl phosphate (CP). (Page 238. Note that ring closing by formation of an amide
bond gives a pyrimidine: 2,4 diketo, 6 carboxyl)
2. Regulatory effectors:
a. inhibited by CTP. Metabolic Relationship (MR) of CTP to E: indirect product - CTP is the
product of the last step in this pathway. (Indirect feedback inhibition)
b. activated by ATP. MR: ATP and CTP are used (along with UTP and GTP) in RNA
synthesis
3. Metabolic Logic of Regulatory Effects
a. When [CTP] is high, ATCase activity to produce more is not needed. Inhibition by CTP
conserves substrates for other uses.
b. When [ATP] is high, RNA synthesis is well supplied ONLY IF [CTP] is also high. Activation
of E by ATP coordinates nucleotide synthesis/supply.
4. Kinetics of E: (v vs. [asp]) The curve (Fig 13-5) indicates positive cooperativity, as with Hb.
The affinity for asp is low at low [asp], and is higher at higher [asp], where several molecules of asp
are bound.
5. Subunit composition: c6r6. The 6 identical catalytic (c) subunits are arranged as two trimers
(one trimer red, one blue in Fig 13-7). When separated from the r subunits, the c’s bind asp and CP
and are active but the kinetics are NOT cooperative. The separated c’s do not bind CTP or ATP, are
not affect by them. The regulatory (r)subunits are three dimers (green in Fig 13-7), which link the c
trimers to each other. They bind CTP and ATP but have no catalytic activity.
6. Molecular Events: Each of the six catalytic subunits has two domains, one of which binds asp
and the other, CP. When substrates bind (one or two pairs, CP + asp), a shift in that/those subunit(s)
occurs in which the two domains move to bring CP and asp together (change in tertiary structure t--->r)
(Fig13-9). This causes the subunits to shift in relation to each other (change in quaternary structure:
T--->R)(Fig 13-7 left-to-right). The catalytic trimers move away from each other and rotate in relation
to each other. More intersubunit interactions are broken than are formed.
B. Summary of changes in interactions and energy (ΔH and so ΔG) which explain kinetic properties
(affinity/cooperativity) of ATCase:
1. T <--->R (When no substrates are bound the T form is favored.) There is a net decrease in
intersubunit (IS) interactions when T--->R (intersubunit bonds broken) ΔHIS is a large positive: (and ΔG
is more positive) for T---> R.
2. T + asp + CP---> asp-R-CP (1st and/or 2nd asp, cp bind)(or 3rd?) There is a gain of
interactions between E and substrates and between substrates, ΔH SB for substrate binding (SB) is a
large negative. The sum ΔGSB + ΔGIS = small (-), low affinity (or at least low rate)
3. asp-R-CP + n CP + n asp---> nasp-R-nCP where n=2? or 3,4,5 or 6. The IS interactions are
already broken, so ΔG = n ΔGSB (and no ΔGIS) so ΔG is large (-) and affinity of R form is high
4. Activation by ATP, which binds strongly only to R form, keeps more E in R form.
R-nATP ---> T + nATP breaks interactions between ATPs and E, so ΔG ATP is +. ΔGIS is - ; sum ΔGATP
+ ΔGIS favors T form less than in 1 above, so more (or most?) of E is in R form. T + CP + asp + ATP
 (cp, asp) – R- ATP (Binding of ATP along with substrates) will have a ΔG ATP that is – to go along
with the – value for ΔGSB, and this will make the overall ΔG more negative and the affinity higher than
in 2 above.
5. Inhibition by CTP (analagous to CO2 effect on Hb) keeps more E in T form, makes T---> R
harder and makes R---> T more favorable; CTP only binds strongly to T. In T-CTP ---> R + CTP,
these are additional interactions that must be broken in T to R. ΔG CTP + ΔGIS for T---> R is more + so
T form is even more favored than in 1, and affinity is even lower than in 2.
6. Effect of the inhibitor “PALA”, a structural analog of (cp-asp). (Binds at active site, competitive
inhibitor)
a. Binds tightly to R form, weakly to T form
b. At “low [PALA]” about one molecule of PALA is bound to each E molecule. PALA binding
“Pays the price” (ΔGIS) for T---> R: ΔGPALA is – for T + PALA-- R-PALA and ΔG(= ΔGIS + ΔGPALA) is
more – than in 1.
c. At “low [PALA]” and “low [asp] and low [CP]” the rate is greater than in the absence of the
inhibitor, because E is in the R form.
VIII. Catalytic Mechanism of an Enzyme
A. Chymotrypsin catalyzes hydrolysis of specific peptide bonds: those proceeded by large
hydrophobic R groups on the substrate.
1. How does E achieve this substrate specificity? Hydrophobic/nonpolar/HC groups on the E
“line” a pocket across from the active site that the hydrophobic group on the substrate fits into and
interacts with.
2. Effects of these interactions:
i. this puts the susceptible peptide bond in extended (β) conformation so that
ii. the peptide N is positioned close to H of ser OH on E and the peptide C is positioned close
to O of ser OH on E. These are “proximity and orientation effects”.
B. Catalytic mechanism of chymotrypsin
1. Step 1: his N accepts H+ from ser OH; this activates ser O to attack peptide C, producing the
first transient tetrahedral intermediate (TS1)
2. Step 2: his N donates H+ to peptide N; activates breaking of peptide bond; producing the
stable acyl – enzyme intermediate and, product 1 is released
3. Step 3: his N accepts H+ from H2O; activates attack of water O on peptide C, producing the
second transient tetrahedral intermediate (TS2)
4. Step 4: his N donates H+ to ser O; activates breaking of E – S bond, releases 2nd product,
returns E to original form
C. Summary of catalytic effects
Note that in each of the transition states, the geometry of the peptide C is tetrahedral and the
negatively charge O atom bound to it is positioned in the “oxyanion hole” where it can H bond to two NH groups of E. The peptide C is trigonal planar at all other times and this O isn’t bonded to E. This is
another example of specific binding of the TS.
Catalytic strategies:
1. proximity and orientation effects
2. acid-base catalysis
3. covalent catalysis (from covalent intermediate with S)
4. specific binding of TS
5. the mechanism of the reaction catalyzed by the E has a different mechanism, involving different
steps. Each of these steps has a much lower activation energy than this reaction has without enzyme.
D. Miscellaneous
1. Serine proteases: a group of related enzymes that use the same catalytic mechanism as
chymotrypsin
2. Trypsin catalyses the same reaction on bonds adjacent to lys and arg side chains: long +
charged groups. It has an asp side chain at the bottom of its specificity pocket that can form an ionic
bond with the substrate side chain.
3. Chymotrypsin is synthesized as an inactive “proenzyme” (to avoid digestion of cellular
proteins). Specific peptide bonds in it must be hydrolyzed to activate it; (catalyzed by other E’s in the
digestive tract).
IX. Enzyme Kinetics is in a separate document, “KmVmax”
X. Enzyme Inhibitors is also in a separate document, “Inhibitors”
XI. Reaction coupling
1. If 2 rxns have a substance in common, they are coupled:
A+XB
∆G = +2.72 kJ
Z+ XY
∆G = -8.42 kJ
2. “X” is the common intermediate by which the two reactions are coupled.
3. To obtain the coupled reaction, one of the reactions may need to be reversed so that “X”
cancels. In order to write the coupled reaction that is spontaneous left to right, one or both reactions
must be reversed so that “X” cancels and the sum for ∆G is negative:
Z+ XY
BA+X
Z+BA+Y
∆G = -8.42 kJ
∆G = -2.72 kJ
∆G = -11.14 kJ