Res. Chem. Intermed., Vol. 29, No. 1, pp.

107– 123 (2003)

Ó VSP 2003.
Also available online - www.vsppub.com

Intramolecularity and enzyme modelling: a critique

SOSALE CHANDRASEKHAR ¤
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India

Received 27 May 2002; accepted 12 September 2002

Abstract—A critical reappraisal of the concept of intramolecularity is attempted, but particulary

focussed on the effective molarity (EM) criterion and the relationship of intramolecularity to enzymic
reactivity. The prevalent ambiguities in the EM concept are addressed and a revised deŽ nition (EMrev )
is suggested. It is argued that there are fundamental limitations to the use of intramolecular reactions
as enzyme models. Although the simplest mechanism for enzymic reactivity is based on transition
state stabilisation, an alternative (although complex) possibility is based on the stabilisation of the
enzyme. Possible mechanisms for the utilisation of the enzymic free energy for effecting catalysis are
discussed.

Keywords: Catalysis; effective molarity; enzyme mechanism; enzyme-modelling; enzyme-stabilisa-

tion; intramolecularity; free energy; Michaelis– Menten; proximity.

INTRODUCTION
That intramolecular reactions are generally different from intermolecular ones has
been known for long. A very pertinent example is that of the ±-hydroxycarbonyl
compound 1, which exists overwhelmingly in the cyclic hemiacetal form 2
(Scheme 1). Since the corresponding acyclic forms are extremely unstable rela-
tive to the carbonyl compound and the alcohol (5, 4 and 3 respectively), the above
equilibrium favouring 2 is a dramatic demonstration of the power of intramole-
cularity to in uence the course of chemical change (see Ref. [1] and references
therein). Although the above equilibrium example demonstrates the thermodynamic
consequences of intramolecularity, the analogous ‘spontaneous’ lactonisation of the
±-hydroxycarboxylic acids (6 ! 7, often requiring no catalyst) (see Ref. [2] and
references therein) demonstrates the kinetic consequences of intramolecularity.
The origins of intramolecularity, however, have not been easy to pin down. The
simplest qualitative approach to understanding intramolecularity adopts the view

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108 S. Chandrasekhar

Scheme 1.

that intramolecular reactions enjoy a relative entropic advantage. Thus, 1 loses

less entropy in forming 2 than do 3 and 4 in forming 5. However, it has not been
easy to quantify this ‘common-sense’ view (as will be discussed further below),
with the result that intramolecularity evolved into a topic of heated debate and
controversy [3 – 9].
The interest in intramolecularity, in fact, derived largely from the belief that it
offered a key to understanding enzymic reactivity [3 – 9]. The advances in the
biochemistry of the proteins made an increasing number of enzymes available
in puriŽ ed form, many of which were analysed by X-ray crystallography. The
fundamental basis of the catalytic power of the enzymes, however, remained a
mystery and thus came under the scrutiny of physical organic chemistry, which
was simultaneously evolving in sophistication. In particular, the success of the
Michaelis – Menten scheme in explaining enzyme kinetics, indicated the existence
of an initially formed enzyme-substrate complex, which produced the Ž nal products
in a series of slow steps (relative to the binding) [9]. Since the rate-determining
reactions subsequent to the intitial binding of the substrate occur intramolecularly
within the enzyme active site, the view gained ground that intramolecularity held
the key to the mystery of enzyme catalysis.
However, it would appear that the prevailing view of intramolecularity needs
to be reassessed, essentially for two reasons. Firstly, the conceptual basis of
intramolecularity itself has to be redeŽ ned, particularly the criterion of ‘effective
molarity’ (EM) and its thermodynamic formulation. Secondly, the link between
intramolecularity and enzymic reactivity seems tenuous vis-a-vis the principles
of transition-state theory. The sheer complexity of enzyme catalysis apparently
demands an alternative and sophisticated rationale. Herein, an elementary but
rigorous thermodynamic approach is adopted in support of the above arguments,
 Intramolecularity and enzyme modelling 109

and to develop an alternative paradigm of enzymic reactivity in consonance with

the accepted principles of chemical reactivity.

DISCUSSION
Effective molarity
Thermodynamic background.
DeŽ nitions and ambiguities. The concept of effective molarity (EM) was pro-
posed as a quantitative measure of the efŽ ciency of an intramolecular reaction rela-
tive to an appropriate intermolecular standard reaction [3 – 9]. Although intramolec-
ularity affects both rates and equilibria, the kinetic consequences were considered to
be more interesting, in view of the belief that intramolecular reactions may model
enzymic ones. Thus, EM was deŽ ned as in equation (1), in which kintra and kinter
are the respective rate constants for an intramolecular reaction and an appropriate
intermolecular one.
EM D kintra =kinter : (1)
There are two problems concerning equation (1): Ž rstly the choice of an intermole-
cular standard reaction is arbitrary; and secondly, since kintra and kinter are unimole-
cular and bimolecular rate constants, respectively, EM retains the dimensions of the
concentration employed in deŽ ning kinter . The latter of the above caveats, particu-
larly, has rendered the EM concept difŽ cult to interpret: thus, EM would vary by
a factor of 103 depending on whether kinter is deŽ ned in terms of mM or M, so the
absolute magnitude of EM seems elusive.
Clearly, the above problem generally arises when rate constants of reactions of
different order are being compared. In fact, this is a manifestation of a general
problem concerning the laws of mass action and of equilibrium (the assumption
in transition-state theory of an equilibrium between the reactants and the transition
state link the equilibrium and kinetic consequences of the above problem). The
fundamental relationship between the equilibrium constant (K) and the standard
free energy change (1G0 ) is given by equation (2). The constant K would
not be dimensionless in the general case but would possess the dimensions of
(concentration)n , n being positive for a dissociative reaction and negative for an
associative one. However, equation (2) is valid as long as the same standard
states are used to deŽ ne both 1G0 and K (alternatively and more rigorously, the
dimensionless activities may be employed to deŽ ne K, but noting that activities
also refer to a standard state [10]). The Eyring relation between the rate constant
‡
(k) and the standard free energy of activation (1G0 ) is given by equation (3) [11]:
1G0 D ¡RT ln K; (2)
¡ ¢
k D .kT = h/ exp ¡1G‡0 =RT ; (3)
¡ ¡ ‡
¢ ¢
exp 1 1G0 =RT D kintra =kinter D EM; (4)
110 S. Chandrasekhar
¡ ¢ ¡ ¢ ¡ ¢
1 1G‡0 D 1G‡0 inter ¡ 1G‡0 intra ; (5)
where T , k and h are the absolute temperature, Boltzmann’s and Planck’s constants,
respectively.
Application of equation (3) to equation (1) above leads to equation (4), in which
1.1G‡0 / is the difference in the standard free energies of activation corresponding
to kintra and kinter as deŽ ned in equation (5). In a way, equation (4) provides a less
ambiguous deŽ nition of EM than does equation (1), as the EM can be referred to
‡
the same standard state as is 1.1G0 /: one notes here that free energy changes are
conventionally referred to a standard state of 1 M, so this serves for EM as well [11].

Towards a new deŽ nition of EM: freedom from standard states.

Relative EM. From the above discussion it is seen that the simple deŽ nition
of EM (equation (1)) is meaningful only when a standard state is deŽ ned by
convention. It is intriguing to consider that this arbitrariness is removed only by
comparing different EMs (all referring to the same standard state). A special case
pertains to the ratio of two EMs of the same class of reaction (say lactonisation),
both referred to the same intermolecular standard: this, of course, is the ratio of the
rate constants for the two intramolecular reactions as seen in equation (6).
EM1 =EM2 D ..kintra /1 =.kinter //=..k intra /2 =.kinter // D ..kintra /1 =.kintra /2 /: (6)
A revised deŽ nition of EM: EMrev . Interestingly, equation (6) offers the opportu-
nity to deŽ ne EM in a novel way, as follows: It is known that the rates of cyclisation
reactions decrease with increasing chain length, until a low steady value for the rate
constant is attained [7] (for instance, in the cyclisation of !-halogenocarboxylates
EM remains steady around 10¡2 for the formation of the 13-membered lactone and
beyond). This value, which persists beyond a certain chain length, may well be
taken as closely approximating the corresponding intermolecular reaction: indeed,
macrocyclisation reactions compete with the corresponding intermolecular ones.
Denoting the macrocyclisation rate constant by kmacro , a revised deŽ nition of EM,
EMrev , is afforded by equation (7).
EMrev D .kintra /=.kmacro /: (7)

Notably, not only is EMrev dimensionless but it is also based on a relatively

unambiguous reference reaction: the macrocyclisation reaction is similar to its faster
analogues than is an intermolecular reference reaction.
It would be trivial, of course, to choose kmacro as one with EM D 1 M (i.e.
kmacro D kinter ) as this merely makes EMrev D EM without the dimensions! It would
be more meaningful to choose kmacro from a genuine macrocyclisation reaction,
based on a fairly steady value for the rate constant. It is seen that macrocyclisation
reactions in general have EM < 1 M: this may be seen as being due to steric and
other constraints that are present in the macrocyclisation transition state but not
 Intramolecularity and enzyme modelling 111

in the intermolecular one — the above discussed caveats about the standard state
remaining [7]. Thus, EMrev > EM (generally, by a couple of orders of magnitude).

Intramolecular reactions as enzyme models

Thermodynamic analysis of intramolecularity.
Gibbs free energy changes. The central problem with the idea that intramole-
cular reactions can serve as enzyme models is the fact that enzyme reactions are
bimolecular. Also, the idea is incompatible with Pauling’s theory of transition state
stabilisation [12], as becomes apparent from a thermodynamic analysis of the EM
concept and the following discussion. Thus, equation (5) may be expanded into
equation (8), where G‡0 and G0 refer to the standard free energy of a transition state
and a ground state, respectively:
¡ ¡ ¢¢ ¡¡ ¢ ¢ ¡¡ ¢ ¢
1 1G‡0 D G‡0 inter ¡ .G0 /inter ¡ G‡0 intra ¡ .G0 /intra : (8)

A high EM implies a substantially lower (1G‡0 /intra relative to (1G‡0 /inter . However,
‡ ‡
a lower (1G0 /intra may result either from a lower (G0 /intra or a higher .G0 /intra
(relative to (G‡0 /inter and (G0 /inter respectively). For the intramolecular reaction to
model the stabilisation of the transition state, a relatively low (G‡0 /intra must obtain.
In fact, not only is there no evidence in support of this, but it can rather be argued
that a relatively high (G0 /intra obtains, as shown below (for simplicity G0 has been
employed in the above arguments, although rigorously the chemical potential needs
to be; these thermodynamic arguments are depicted in Fig. 1) [13].
Entropy changes. Equation (9) essentially derives from equation (8) by substi-
tuting the negative entropy (–S/ for free energy (G/ and rearranging.
¡ ¡ ‡ ¢¢ ¡¡ ‡ ¢ ¡ ¢ ¢
1 1S0 D S0 intra ¡ S0‡ inter ¡ ..S0 /intra ¡ .S0 /inter /: (9)

This is based on the deŽ nition 1G‡0 D 1H0‡ ¡ T 1S0‡ , where H is the enthalpy.
A relatively large (1.1S0‡ // would favour the intramolecular process, and this
‡ ‡
may arise either from a relatively large ..S0 /intra ¡.S0 /inter / term or a relatively small
..S0 /intra ¡ .S0 /inter / term: notably, both these terms would generally be negative as
there is a relative loss of translational entropy corresponding to one molecule in the
intramolecular model.
Clearly, therefore, a favourable (1.1S0‡ // would depend on the entropic cost of
intramolecularity being less in the transition state than in the ground state, i.e. the
‡ ‡
((S0 /intra ¡ .S0 /inter / term being less negative than the ((S0 /intra ¡ .S0 /inter / term
(as depicted in Fig. 1). Indeed, the transition state of a bimolecular reaction has
essentially no translational freedom to lose if (hypothetically) it is converted into
a cyclic transition state: both the inter- and intramolecular transition states consist
of a single molecule, and so their translational entropies would be similar. The
intramolecular and intermolecular ground states, however, are expected to be vastly
112 S. Chandrasekhar

Figure 1. Thermodynamic consequences of intramolecularity. The left hand side represents the
intermolecular reaction between A and B, and the right hand side the corresponding intramolecular
version. The lower half represents the ground states and the upper half the transition states. H 0 (- - -),
S0 (¢ ¢ ¢) and G0 (—) represent enthalpy, entropy and Gibbs free energy respectively at the standard
state. Transition states are represented by ‘‡’ and a partial bond in them by a relatively long dotted
line (¢ ¢ ¢). The entropic cost of intramolecularity is greater in the ground state than in the transition
state: thus .1G0 / > .1G 0 /‡ and the cyclic transition state is ‘relatively stabilised’. The cyclic species
are characterised by relatively high enthalpy and low entropy. Although enthalpic effects are assumed
to be relatively small, relief of strain in the cyclic transition state would mean that .1H0 / > .1H0 / ‡
(not indicated). The depiction is qualitative and not to scale.

different entropically, with the latter beneŽ tting from the extra translational entropy
corresponding to one molecule.
Similar qualitative considerations would apply also to vibrational entropy, which
is expected to decrease more markedly in the ground state than in the transition
state, in the intramolecular case relative to the intermolecular: proximity results
in restricted conformational freedom and a consequent decrease in vibrational
entropy, but most of such restrictions are common to both the intermolecular and
intramolecular transition states.

Enthalpy changes. The enthalpic contribution to EM is difŽ cult to predict.

Generally, high EMs may well derive from relatively low enthalpies of activation
‡ ‡
(1H0 / acting in concert with the above discussed entropic effects. Low 1H0 values
may again derive from either high ground state enthalpies (H0 / or low transition
‡
state enthalpies (H0 /, relative to the intermolecular case. However, it appears likely
that high H0 contributes signiŽ cantly to high EM, as intramolecular reactions can be
driven by relief of ground state strain: indeed, steric and other strain are generally
 Intramolecularity and enzyme modelling 113

expected to increase when the reactive groups are brought into proximity (with a
consequent decrease in entropy as discussed above).

Pros and cons of intramolecularity.

High EM: a ground-state phenomenon. The picture that emerges from the above
thermodynamic discussion is one of intramolecular reactions being dominated by
ground-state effects: high H0 derived from steric and other strain as a consequence
of enforced proximity, and low S0 derived from restricted translational and vibra-
tional freedom. The resulting high G0 appears to be the major contributor to the
relatively low 1G‡0 of intramolecular reactions (Fig. 1). There is no evidence for
the absolute stabilisation of the cyclic transition state, so clearly intramolecular re-
actions are inappropriate models for enzyme catalysed reactions.
Relative stabilisation of the cyclic transition state. However, high EMs can be
viewed as being derived from a relative stabilisation of the intramolecular transition
state: this is less destabilised (particularly entropically) than is the intramolecular
ground state — relative to the corresponding intermolecular states — as argued
above. Also, it is erroneous to believe that the intramolecular transition state is more
stable than the intermolecular analogue because of the extra bonding that confers
intramolecularity: the ‘linker bond’ in the cyclic case would be replaced by two
other bonds in the intermolecular case (quite apart from the fact that the ‘linking’ is
equally present in the cyclic ground state).
The above arguments are represented in cartoon fashion in Fig. 2. The hypothet-
ical reaction of the reactive centres A and B may be either inter- or intramolecular,
and the key question in the present context is: is the cyclic transition state (TScycl )
more or less stable than the acyclic one (TSacycl )? In fact, both these species may
be modelled by stable analogues in an attempt to answer the above question; e.g.
cyclohexane may be considered as an analogue of TScycl and n-hexane as that of
TSacycl (the partial bonds in the transition states are replaced by C C bonds in the
analogues).
Interestingly, n-hexane is more stable than cyclohexane as shown by their
respective standard Gibbs free energies of formation (1G0 D ¡0:91 versus
C6:37 kcal/ mol) [14, 15]. This clearly indicates that cyclic transition states are
not generally more stable than their acyclic analogues (as already mentioned, even
macrocyclisation reactions often have EM < 1, thus indicating that the correspond-
ing TScycl are not relatively strain-free). As a general rule cyclic compounds are
destabilised entropically by restricted conformational freedom; enthalpically too
they are often destabilised by various types of strain (the 1H0 values for the forma-
tion of n-hexane and cyclohexane are ¡47:3 and ¡37:2 kcal/ mol, respectively; the
respective entropy contents S0 are 69.2 and 64.3 cal / K per mol [14, 15]).
Thus, there is no evidence to support the assumption that the ‘linker bonds’
(solid lines in Fig. 2) bestow any stability on TScycl : the effects of such linking
on the partial bond in TScycl would be similar as on the full bond in the model
114 S. Chandrasekhar

Figure 2. A comparison of an intermolecular reaction between the reactive centres A and B (left)
with the corresponding intramolecular analogue (right), to produce their respective transition states
TSacycl and TS cycl . The C H bonds in the intermolecular case are replaced by C C bonds in
the intramolecular case: so there is no ‘additional binding’ in TScycl despite the ‘linker bond’
(solid). TSacycl and TScycl may modelled by an open chain and a cyclic compound respectively:
TScycl generally strained relative to TS acycl indicating that the origins of intramolecular and enzymic
reactivity are fundamentally different. See Fig. 1 for a thermodynamic analysis of the above set.

cyclohexane (i.e. essentially destabilising as seen above). On the basis of the above
arguments, therefore, it seems highly unlikely that a lowered TScycl is the origin of
intramolecular reactivity.

Pseudo-unimolecularity, enzyme catalysis and intramolecularity. The idea that

intramolecular reactions serve as enzyme models essentially derives from the fact
that enzyme catalysed reactions occur via two major steps, an initial binding step
followed by the actual catalytic step (which is composed of several relatively slow
steps). The important catalytic step is clearly intramolecular in that it occurs within
the active site cavity of the enzyme. Whilst there is no question that appropriate
intramolecular analogues would model this step, they would not model the overall
enzyme-catalysed reaction, which includes the Ž rst binding step.
An important feature of enzyme catalysis is that of saturation kinetics, by
which the rate of the enzymic reaction approaches a maximum value (Vmax ) at
relatively high substrate or enzyme concentration [9]. At relatively high substrate
concentrations the Michaelis – Menten equation (MME) takes the form Vmax D
kcat [E0 ], kcat being the turnover number and [E0 ] the total enzyme concentration.
Thus, the saturation regime exhibits pseudo-unimolecular kinetics: interestingly,
because of the original kinetic form of the MME derived from the pre-equilibrium
binding of the substrate with the enzyme, the substrate concentration ‘drops off’ and
the rate depends only on [E0 ]. As [E0 ] » [ES] (the concentration of the enzyme-
substrate complex) at saturation, the overall enzymic reaction appears to be just the
 Intramolecularity and enzyme modelling 115

unimolecular reaction of ES. This seems to justify the use of intramolecular models
for enzymic reactions.
However, the problem with the above-mentioned approach is that the kinetic
features of a reaction under pseudo-molecular conditions can be misleading: in
particular, the temperature coefŽ cient of the rate constant thus obtained will not
provide the real free energy of activation of the overall process [16]. In the case
of the MME the saturation regime creates the impression that the formation of
ES is of no consequence: whilst the kinetic form apparently justiŽ es this, it is
misleading thermodynamically. (A recent treatment [3], in fact, essentially states
that the entropic requirements of an enzyme catalysed reaction are similar to
those of its non-catalysed analogue, but by comparing kcat with the rate constant
of the non-catalytic reaction! Apparently, this leads to the questionable view
that the binding of ‘near attack conformers’ rather than of the transition state is
important.)
A thermodynamic analysis of enzyme catalysis has been presented previously [17].
Essentially, the efŽ ciency of enzyme catalysis derives from the efŽ ciency of both the
binding and the turnover steps. The binding can be either endergonic or exergonic,
but in either case, any additional binding energy can be carried over advantageously
to the transition state. In the present context, however, the fact remains that the rate
determining transition state in the overall enzymic reaction is essentially a bimole-
cular one, as the reference ground state consists of the free (unbound) enzyme and
the substrate. And the fact that the enzymic reaction becomes intramolecular along
the way (i.e. after the binding of the substrate) is not germane to the argument as,
according to the transition-state theory, the dynamics by which the transition state
is reached are thermodynamically insigniŽ cant [13].

Proximity: a ‘consequential necessity’. On the above bases, the binding of

the substrate close to a reactive group on the enzyme does not indicate a simple
proximity effect. Whilst it is true that the substrate must bind within ‘striking
distance’ of a reactive group on the enzyme to react at all, the relation between
reactivity and proximity should strictly be interpreted in terms of transition state
effects. Thus, the transition state would be less strained when the substrate is
bound close to the reactive group rather than at a distance. Therefore, proximity
in the usual sense is not the cause of enzymic reactivity: rather, proximity is a
‘consequential necessity’, i.e. it is a consequence of the necessity that the enzyme
binds the substrate, and in a way that ensures its unhindered progression to the
bound transition state, the key species (noting that ‘remote binding’ is ineffective).
Thus, proximity between the reactive groups on the substrate and on the enzyme
is necessary, but not sufŽ cient, for high reactivity: the only sufŽ cient condition
is the stabilisation of the transition state in some way. Clearly, the deŽ nition of
proximity in the enzymic case is rather subtle, and is different from that in the case
of intramolecular reactivity.
116 S. Chandrasekhar

Enzyme models
Present scenario. The preoccupation with proximity and intramolecularity may
well have hindered alternative approaches to understanding enzyme catalysis. At
the very least, the Pauling hypothesis of transition state stabilisation (in the absolute
sense) could have been pursued more vigorously than it was. Intramolecular reac-
tions cannot generally attain the rates of bimolecular enzyme catalysed reactions
[5] (!): this should make it abundantly clear that enzymes are special and invoke
mechanisms that are not easily modelled in simple systems.
In the following parts novel approaches to enzyme catalysis are presented in
a general way. Stress is laid on the fundamental principles and concepts in
order to clarify the novel arguments presented. Their detailed elaboration and
quantitative formulation are beyond the scope of the present treatment. However,
mechanistic enzymology is well served by several masterly reviews and books (see
Refs [9, 16, 18]), which would enable a comparison of the present treatment vis-a-
vis the existing formulations.

The hydrophobic effect and transition state stabilisation. It is only relatively

recently that the possible molecular details of the Pauling hypothesis have been
addressed [19– 21]. However, even the hypothesis of transition-state stabilisation
may be an oversimpliŽ cation, as the binding of the substrate with the enzyme
is apparently driven by the hydrophobic effect [9]. This essentially implies
that the relative destabilisation of the substrate in aqueous solution provides a
signiŽ cant part of the required activation energy (this is discussed again below
under Intramolecular models). The hydrophobic energy may be used to distort the
substrate towards the transition state at the enzyme active site. It then remains
for the charges developed at the transition state to be transmitted away from
the hydrophobic interior, towards the aqueous exterior by an appropriate relay
mechanism (the relay could also provide additional binding of the transition state).

Substrate distortion. Any distortion of the substrate per se by the enzyme,

however, cannot be the origin of the catalysis: the binding of the distorted substrate
needs to be carried over to the transition state. Generally, the distortion of the
substrate by the enzyme — hydrophobic effect or not — is disadvantageous as it
results in a high Km (the Michaelis constant: it has been argued that low Km and
high kcat are both required for efŽ cient enzyme catalysis [17]). However, if the active
site is exclusively complementary to the transition state, the substrate can only be
bound in a highly distorted state: clearly, a balance between the binding of the
substrate and the binding of the transition state needs to be struck (transition-state
complementarity of the active site increases kcat at the risk of a high Km , whereas
substrate complementarity decreases Km but at the risk of a low kcat . It would, of
course, be ideal if the enzyme could bind both the substrate ground state and the
transition state, effectively supplementing the ground state binding at the transition
state [17]).
 Intramolecularity and enzyme modelling 117

The apparently contradictory consequences of substrate distortion above, may be

clariŽ ed by viewing the phenomenon as providing a means for the utilisation of the
hydrophobic energy to approach the transition state. However, the activation energy
barrier cannot be ‘offset’ in any way by the hydrophobic effect in the ground state:
the barrier can only be reduced by stabilising the transition state, at which point the
maximum hydrophobic (and other) binding would be expressed.
Intramolecular models. Intramolecular reactions, of course, model the transfor-
mation of the enzyme-bound substrate to the transition state. It is tempting to view
intramolecular reactions, particularly those driven by relief of strain, as evidence
in support of the substrate distortion theory of enzyme catalysis. Thus, it may be
argued that the hydrophobic effect imposes — via the formation of the enzyme-
substrate complex — proximity between the reactive groups of the substrate and
the enzyme, or that the substrate is bound in a strained conformation. However,
such approaches  ounder against the principles of transition state theory, which un-
compromisingly require the transition state to be stabilised in the enzymic case.
It may similarly be argued that the hydrophobic effect leads to a raised ground
state which manifests as strain, etc., at the enzyme active site, and that intramole-
cular reactions are an appropriate model for this dynamic. However, this is only
apparent as — in terms of reactivity — the raising of the ground state is relative to
the transition state, which has been stabilised by the enzyme. Thus, the origins of
the reactivity are different in the intramolecular and enzymic cases.
Proximity and enzyme catalysis. The above arguments do not mean that prox-
imity has no role to play in enzymic reactivity. An important feature of enzyme
reactions is multifunctional catalysis: the active sites of enzymes consist of several
catalytic groups (mild general acids and bases, co-enzymes, etc.), which have been
spatially organised in an optimal and effective fashion by the folding of the protein
back-bone. Clearly, the interactions between these catalytic groups is intramolecu-
lar: the entropic requirements of the corresponding intermolecular ensemble would
indeed be enormous. The arguments against intramolecularity presented above, thus
pertain only to the interactions between the enzyme and substrate.

Future prospects.
The enzyme molecule at centre-stage. The theory of transition state stabilisation
essentially focusses on the substrate molecule, particularly the changes it undergoes
at the transition state, with the enzyme molecule playing an as yet ill-deŽ ned role
in stabilising the transition state. An alternative and interesting approach would
instead focus on the enzyme molecule and the changes that it undergoes during
the passage to the transition state. Clearly, the enzymic reaction involves both the
substrate and the enzyme, so the activation energy would re ect changes in both
of them. In fact, an elementary thermodynamic treatment of the problem leads to
interesting insights, but Ž rst an important clariŽ cation would be in order. A recent
treatment [3] interestingly assumes the same ground state energy for an enzyme
118 S. Chandrasekhar

catalysed reaction as for the non-catalysed analogue! The paper also concludes
generally that enzymes do not stabilise the transition state either!
Enzyme catalysis and molecularity. As an enzyme catalysed reaction is kinet-
ically of higher order than the uncatalysed analogue, their mutual comparison is
also bedevilled by the same complications as is the intramolecularity problem dis-
cussed earlier. Thus, the ratio kuncatalysed = kcatalysed would possess the dimensions of
(concentration)n where n is the order of the reaction in the enzyme: the magnitude
of this ratio would thus depend on the choice of the standard state for the enzyme.
In the present context, it is also interesting to deŽ ne a concentration dependent
(but dimensionless) rate constant kobsd as kobsd D [E] £ kcatalysed , where [E] is
the concentration of the enzyme. Then the rate ratio r would be deŽ ned as in
equation (10). The above arguments have an important bearing on the general theory
of enzymic reactivity as will be clariŽ ed later.

r D kuncatalysed =kobsd D kuncatalysed =.[E] £ kcatalysed /: (10)

Gibbs free energy composition of enzyme catalysed reactions. The standard free
‡
energy content of the transition state of an enzyme catalysed reaction (G0 /ES would
be composed of contributions from both the enzyme (G‡0 /E and the substrate (G‡0 /S
as in equation (11), the corresponding ground state being represented analogously
in equation (12).
¡ ‡¢ ¡ ¢ ¡ ¢
G0 ES D G‡0 E C G‡0 S ; (11)
.G0 /ES D .G0 /E C .G0 /S ; (12)
¡ ‡
¢ ¡¡ ‡ ¢ ¢ ¡¡ ‡ ¢ ¢
1G0 ES D G0 E ¡ .G0 /E C G0 S ¡ .G0 /S ; (13)
¡ ¢ ¡ ¢ ¡ ¢
1G‡0 ES D 1G‡0 E C 1G‡0 S ; (14)
©¡ ‡
¢ ¡¡ ‡ ¢ ¢ ¡ ‡
¢ ¡¡ ‡ ¢ ¢ª
1G0 E D G0 E ¡ .G0 /E and 1G0 S D G0 S ¡ .G0 /S ;
¡ ¢ ¡ ¢ ¡¡ ¢ ¢
1G‡0 UN D 1G‡0 S;UN D G‡0 S ¡ .G0 /S UN; (15)
¡ ¢ ¡¡ ¢ ¡ ¢ ¢ ©¡ ¢ ª
1 1G‡0 D 1G‡0 ES ¡ 1G‡0 UN D G‡0 ES ¡ ..G0 /E C .G0 /S /
¡¡ ¢ ¢
¡ G‡0 S ¡ .G0 /S UN D ¡.G0 /E ;
¡ ¡ ‡¢ ¡ ¢ ¢
as G0 ES D G‡0 S ; (16)
¡ ¢ ¡¡ ¢ ¡ ¢ ¢ ¡ ¢ ¡¡ ¢ ¡ ¢ ¢
1 1G‡0 D 1G‡0 ES ¡ 1G‡0 UN D 1G‡0 E C 1G‡0 S ¡ 1G‡0 S;UN : (17)

Equations (13) and (14) represent the free energy of activation for the enzyme
catalysed reaction (as a sum of the contributions of the enzyme and the substrate);
and equation (15) represents that for the corresponding uncatalysed reaction (the
‡
term (1G0 /S;UN indicates that only the substrate is involved). Equations (16)
and (17) represent, in different ways, the difference in the free energies of activation
between the enzyme catalysed and uncatalysed reactions (1.1G‡0 //; both these
relations lead to interesting insights as discussed further below.
 Intramolecularity and enzyme modelling 119

Equation (16) decomposes the difference in the free energies of activation between
the enzyme catalysed and uncatalysed reactions into the contributions of the
corresponding transition states and ground states. If it is assumed that the free
energy contents of the transition states of both the reactions are the same, i.e.
‡ ‡
(G0 /ES D .G0 /S , it is seen that the free energy of activation of the enzymic
reaction is lower by an amount equal to the free energy content of the enzyme
((G0 /E ). Equation (17), on the other hand, decomposes the difference in the free
energies of activation between the enzymic and non-enzymic reactions (1.1G‡0 //
into the contributions from the enzyme and substrate parts: this, in fact, leads to the
remarkable concept of the stabilisation of the enzyme.
The enzyme as a free energy reservoir. As seen above equation (16) leads to the
possibility that the enzymic acceleration derives from the free energy contribution of
the enzyme. Clearly, the enzymic acceleration may be attributed either to a lowered
transition state or to a raised ground state: the Pauling theory is based on the former
possibility, but the latter offers an interesting alternative model that is discussed
in the following sections (it is also noteworthy that the above two mechanisms
represent extreme possibilities and that both may be involved in practice).
It was noted above that the magnitude of the ratio kuncatalysed = kcatalysed for an
enzymic reaction depends on the standard state employed for the enzyme: this
clearly implies that the free energy of activation in the enzymic case depends on
the free energy contribution of the enzyme. Equation (10) makes it obvious that the
ratio of the rates (r) depends on the concentration of the enzyme employed: this
result can also be realised rigorously by employing the chemical potential (¹) but
will not be repeated here [17].
The key assumption above is that the catalysed and uncatalysed transition states
are equal in free energy content. This possibility may be appreciated qualitatively
by invoking a transfer of free energy from the enzyme to the substrate, to raise the
latter to the transition state upon binding: in the ideal case the free energy content
of the enzyme matches the free energy of activation of the uncatalysed reaction.
This unusual mechanism requires the free energy content of the enzyme (G0 /E to be
relatively high and positive: therefore, although it is apparently viable in principle
it would apply only when (G0 /E is demonstrably high. This possibility is discussed
in some detail later, but a variant based on equation (17) is described below.
The stabilisation of the enzyme at the transition state. For effective catalysis by
the enzyme the term 1.1G‡0 / in equation (17) must be negative. If it is assumed
that the free energy changes in the substrate part are the same in the catalysed
‡ ‡ ‡
and uncatalysed reactions, i.e. ((1G0 /S ¡ .1G0 /S;UN/ D 0; 1.1G0 / can only be
negative if (1G‡0 /E is also negative. This can only mean that the enzyme is stabilised
at the transition state of the reaction, and by an amount equal to the lowering of
the free energy of activation of the reaction by the enzyme (since (1G‡0 /E now
represents this). This, then, is an alternative thermodynamic formulation of the
mechanism of enzyme action, to that of the stabilisation of the transition state: it
120 S. Chandrasekhar

is a possible way in which the free energy of the enzyme molecule is utilised for
effective catalysis (the stabilisation of the enzyme at the transition state has indeed
been proposed before, see pp. 7 and 8 in Ref. [6]).
Stabilisation of the transition state versus transfer of free energy. The mecha-
nism of enzyme action presented above apparently differs from the Pauling mech-
anism based on transition state stabilisation. However, the present mechanisms es-
sentially focus on the modes of energy transfer between the enzyme and the sub-
strate. Essentially, a  ow of free energy from the enzyme to the substrate is being
proposed, with the enzyme being stabilised at the transition state (either absolutely
or relatively). Of course, the enzyme-bound transition state is a single molecular
entity, and the distinction between the substrate part and the enzyme part is arbi-
trary. However, two extreme cases may be envisaged in which either the enzyme
or the transition state (relative to the uncatalysed path) undergoes major structural
changes. These would indicate, respectively, the stabilisation of either the enzyme
or the transition state, with intermediate mechanisms also being possible.
It is noteworthy that the stabilisation of the transition state requires that work
be performed by the enzyme on the transition state (by dissipating charges, etc.),
which in effect requires at least a part of the free energy content of the enzyme
to be released (for the performance of the work). Although ‘work’ is a concept
of macroscopic thermodynamics and is not easily deŽ ned at the molecular level,
the above arguments apparently indicate that the present proposals may not be
fundamentally different from the original Pauling hypothesis.
The free energy content of an enzyme. In general, enzymes are relatively large
protein molecules. Interestingly, the free energy contents of polypeptides are gen-
erally higher than the total free energy contents of the component amino acids [22].
The ¡1H0 (kcal/ mol), ¡1S0 (cal / K per mol) and ¡1G0 (kcal/ mol) values, re-
spectively, for the formation of glycine and some of its oligomers are: glycine 128.4,
127.9 and 90.3; glycylglycine 178.1, 206.5 and 116.6; di-glycylglycine 230.1, –,
145.1; tri-glycylglycine 283.7, –, 175.3. Thus tri-glycylglycine is unstable with re-
spect to six molecules of glycine by a 1G0 of 366.5 kcal/ mol, etc. This seems to
indicate that the larger proteins may possess relatively high — perhaps positive —
free energy contents. However, it is not necessarily the absolute free energy con-
tent of the enzyme that is critical: rather, it is important for the enzyme to be able
reversibly to access a lower energy form during the catalytic reaction, and in the
process surrender free energy to the substrate and subsequently recoup it after the
transition state of the reaction is passed (cf. equation (17) and the stabilisation of
the enzyme).
Reversible energy transfer between substrate and enzyme. A possible mecha-
nism for the above process of reversible energy transfer from the enzyme to the
substrate is based on the enzyme’s being able to access an alternative conforma-
tional form, the two forms being separated by an activation energy barrier. A protein
molecule, of course, has an enormous number of conformational states available to
 Intramolecularity and enzyme modelling 121

it (derived largely from restricted rotation around the peptidic amide bonds) [23, 24],
and need not exist in the most stable of these forms. The native protein would adopt
the form that is most preferred during the conditions extant during protein synthesis
at the ribosomal site: this form would then persist if the conversion to other forms
(though more stable they may be) is very slow, i.e. the above form is protected by
relatively high energetic barriers.
Thus, the alternative form of the enzyme would be accessible if there is an input
of energy sufŽ cient to overcome the conformational energy barriers separating the
two forms. The energy released during the conformational transition could be ‘fed’
to the bound substrate molecule, ‘helping it over the barrier’: once this is crossed,
the substrate molecule could release the energy back to the enzyme during the slide
to the newly formed product molecule. The process thus consists of an intial input
of energy to the enzyme molecule to overcome the barrier to the conformational
transitions, followed by the release of this activation energy along with the energy
of the conformational transition to the substrate molecule.
The missing piece in the above puzzle is the origin of the initial energy that is
needed to overcome the barrier to the conformational transition: it is interesting to
consider this to be the energy released upon the formation of the enzyme-substrate
complex (ES)! On this basis, the formation of ES is per se exergonic: however,
the energy of formation of ES is not released to the surrounding environment but
is retained in the enzymic moiety. Thus, the formation of ES coincides with the
crossing of a conformational energy barrier in the enzymic moiety (A in Fig. 3), and
the crossing of the energy barrier of the reaction involving the substrate coincides
with the end of the conformational transition (at E0 in Fig. 3). Conformational
changes in enzymes induced by the binding of the substrate are indeed known
(e.g. the phenomenon of allosteric control) [25], so the above mechanism is not
improbable.
In fact, yet another related mechanism for the stabilisation of the enzyme is
interesting. The fact that a proteinic enzyme molecule exists in a relatively Ž xed
conformation despite the possibility of a very large number of states, indicates a
corresponding sacriŽ ce in entropy relative to the denatured (‘random coil’) state
of the protein, and a correspondingly high free energy content (estimated to be
5 – 15 kcal/ mol) [24]. If there is a substantial loosening of the proteinic back-bone
of the enzyme (approaching the random coil state) during the catalytic reaction, it
may be envisaged that a substantial part of the ‘entropic free energy’ of the enzyme
could be channelled productively as discussed above.
Clearly, however, all such mechanisms involve novel patterns of redistributing
energy (presumably vibrational) within the enzyme-substrate complex: such ‘non-
ergodic’ models have indeed been proposed but have been controversial (see
Ref. [26] and references therein). However, in view of the above arguments that
the complexity of enzyme catalysis may have been underestimated, such alternative
possibilities may need to be reconsidered (for an elegant appraisal of the complexity
of enzyme catalysis, see Ref. [27]).
122 S. Chandrasekhar

Figure 3. Possible changes in the Gibbs free energy (G) in the substrate (S, solid line) and in the
enzyme (E, dashed line) during the enzyme catalyzed conversion of S to product P. The energy
released upon the formation of the enzyme-substrate complex (ES) provides the activation energy
for the enzyme to effect a conformational transition to an alternative form (E0 ) by crossing the barrier
at A. The energy released during the slide from A to E 0 provides part of the activation energy for
the formation of the transition state ES‡ . Similarly, the energy released during the formation of
the enzyme-product complex (EP) is used to effect the reconversion of E0 to E, which is released
Ž nally along with P. The relative stabilities of E and E0 need not be as shown (and E0 cannot be
released from ES ‡ ). The solid and dashed proŽ les are shown at different levels only for convenience
of representation.

CONCLUSIONS
A revised deŽ nition of effective molarity, based on an appropriate macrocyclisation
reaction as a reference standard, provides a set of ‘EMrev ’ values that are dimen-
sionless and relatively unambiguous. However, the relevance of intramolecularity
to enzymic reactivity is tenuous as the enzymic reaction is essentially bimolecular,
the origins of the reactivity being different in the two cases. Thus, intramolecularity
beneŽ ts from a ‘raised ground state’ (derived from low entropy and high enthalpy)
whereas the enzyme reaction needs to be stabilised at the transition state by the
Pauling hypothesis. An interesting alternative to the Pauling mechanism, however,
involves a  ow of free energy from the enzyme to the substrate and an effective
stabilisation of the enzyme at the transition state: enzymic reactivity, thus, is appar-
ently more complex than previously believed.

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