TDC–October, 2020

Enzymes as machines
Introduction

Contacting Lecturer
Use the Discussion Group link – this is the most efficient (other students often get there sooner, and might
be better at diagnosing a problem that they themselves have just solved. I will always check and correct if
need be.) Note that you can choose to be anonymous.
If there is something that can not go on a discussion board, E-mail: Paul.McLaughlin@ed.ac.uk

References
 Alberts, BM, et al. Molecular Biology of the Cell. 5th edition. Garland Science, New York, pp 159-169
 Berg JM, Tymoczko JL, Stryer L Biochemistry 5th Edition WH Freeman New York, Chapter 8, p435

Aims
You should be able to:
 illustrate how an enzyme is not an energy consuming machine but rather a molecular ‘jig’ that
facilitates the reaction it catalyses;
 describe how a phosphoryl-enzyme intermediate can be ruled out in the mechanism of
phosphoglycerate kinase;
 list the results that show direct transfer of phosphate between substrates, bound at two separate sites, is
consistent with the mechanism of the enzyme;
 describe the evidence that the leaving phosphate group is attacked directly in-line with the bond that is
broken;
 illustrate how the enzyme stabilises the transition state;
 show how the enzyme protects the substrate from hydrolysis and why this is important;
 argue that free energy is released on the enzyme not when product is formed but when it is released
from the enzyme; and
 discuss the evidence that substrates are channelled to the enzyme from the preceding enzyme in the
glycolytic pathway.

Overview
The cell is not just a membrane bounded bag in which processes occur by blind diffusion only. Through the
agency of proteins (see Block 1), small metabolites are processed and thereafter handed on to another
protein, often in the same complex, without diffusion in the crowded cytoplasm being necessary. An
enzyme involved in glycolysis , phosphoglycerate kinase, is an appropriately simple system that allows us
to appreciate the role of protein in orchestrating the different sort of chemistry necessary for cell function.
We will establish some useful principles regarding phosphate transfer to and from ATP, which will aid our
understanding of motor proteins and pumps (see later lectures in this block).

Enzymes as molecular ‘jigs’

To call an enzyme a molecular machine is not strictly an appropriate analogy. A machine transduces energy
to do work and ends up in a different state at the end of the process. An enzyme facilitates a reaction but
remains unchanged at the end.

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A better analogy, again from engineering, is to

describe an enzyme as a jig that is a frame into
which components are assembled to make them
easier to work on. We shall see that protein forms
this frame and aligns the reactants so that the
reaction takes place more easily. At the same time
components are protected from ‘spoilage’ by other
agents in the workshop (the cell). When the
completed product is removed, the “jig” is
unchanged. New components can be loaded onto it
for the next cycle of production.

Phosphoglycerate Kinase – Reaction and Biological Context

Phosphoglycerate kinase catalyses a
relatively simple reaction in which a
phosphate group from 1, 3
diphosphoglycerate (1, 3 PGA) is
transferred to ADP, leaving 3
phosphoglyceric acid (3PGA) and ATP.
As such, the enzyme catalyses the first
ATP producing step in glycolysis (forward
direction).

Investigating the mechanism

Kinetics of the enzyme-catalysed reaction

By studying the kinetics we can show that the enzyme
directly transfers the phosphate group to ADP as shown in
the diagram. This proposes that both substrates can bind
simultaneously, and the order in which they come on or off
doesn’t matter.

If there are separate sites for ADP and 1, 3 PGA and we

saturate the ADP site, the enzyme behaves as if it were a
simple enzyme that had only one site for 1,3PGA. As we
vary the concentration of 1,3PGA, and plot its rate of
disappearance, we would observe the usual hyperbola we
expect for a single site enzyme (right). We should also see
hyperbolic kinetics if we reversed the order and saturated
with 1,3PGA but varied ADP.

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This behaviour rules out one other possible mechanism, that in which the phosphate is transferred to the
protein and then ADP picks it up: that mechanism would require only one binding site but a defined order in
which the reactants bind.

In fact, when the experiments are done, the order of which substrate saturates and which is varied does not
matter and both experiments show hyperbolic kinetics at saturating concentrations of the other substrate.
This is inconsistent with the phosphoryl-enzyme mechanism. It is consistent with the direct transfer
hypothesis but does not prove it.

Phosphate transfer occurs by in-line attack with inversion of configuration

In many phosphoryl transfer reactions, the leaving phosphate is

attacked in-line with the bond that is to be broken. In that case
there is an inversion of the configuration of the oxygen groups
around the phosphate group that is hydrolysed.

The experiment is consistent with in-line attack of the leaving phosphate of 1,3 PGA by ADP. The
consequence is that both the leaving and attacking phosphates must be very precisely aligned. In solution
the chances that this would happen by random diffusion are much more limited, so the uncatalysed reaction
is much slower.

Principles of catalysis employed by the enzyme

Reduce activation energy of transition state

A consequence of in-line attack and inversion of configuration of
the leaving phosphate group is that the phosphoryl oxygens pass
through a high-energy state. This is shown to the right. As the
ADP phosphate attacks, a pentavalent intermediate forms at the
leaving phosphate. Three of its oxygen groups are in a plane,
while the attacking oxygen and leaving oxygen are perpendicular
to the plane. The oxygens in the plane are negatively charged and
are much closer to each other in this configuration than they are
before or after the reaction. Thus this transition state is a high-
energy state. Passing through this high-energy state limits the rate
of the uncatalysed and the enzyme catalysed reaction. But the
enzyme speeds the reaction because it can stabilise the transition
state. It does so with positively charged groups on the enzyme.

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The positive groups on the enzyme are Lysine 219 and Arginine
39 (Trypanosoma brucei PGK). The figure is a composite of
several crystal structures of PGK in the presence of different
components of the reaction. The phosphate group between ADP
and 3PGA is close to the position of the leaving phosphate as it
transits from 1,3PGA to ADP. In the structures, both Lys 219
and Arg 39 are within hydrogen bonding distance of the
transferring phosphate group. Notice the bound Mg2+ ion (often
a physiological counter-ion for ATP). The positive charges on
this ion also stabilise the transition state.

Protect from side-reactions

Perhaps as important as speeding up the reaction,
PGK promotes phosphate transfer to ADP over
transfer to water. In fact 1,3 PGA, as a phosphate
ester, is very unstable in water. It is easily
hydrolysed, whereupon a phosphate is released.
Unlike ATP, this phosphate cannot be further
hydrolysed to release energy that can be put to work
in the cell. If hydrolysis were to occur, one of the
ATPs from glycolysis would be lost. The danger is
that the enzyme would stabilise the transition state
for the hydrolysis reaction just as well as that for
transfer to ATP. It would be best to shield the
substrates from water, but this is difficult because
the substrate binding sites must be quite open since
the substrates need to gain access from bulk solution.

Crystal structures showed how the enzyme solves this

problem. The protein is folded into two domains.
3PGA binds to one domain, which encompasses the
N-terminal half of the molecule. The other substrate
binds to the C-terminal half domain. These sites are
too far apart to be in direct contact and for the
phosphate to be transferred between them. It took
many years of work to grow crystals of PGK with
both substrates. The resulting structure showed a large
hinge bend between the domains that positioned the
substrates closely together and buried them away from
water. Moreover, Lys 219 is on the C-terminal half
and Arg 39 on the N-terminal domain. These residues
only come into position to stabilise the transition state
when both substrates bind.

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PGK reaction in cells – products only transiently leave enzyme

Consider the following concentrations of PGK, GAPDH , 1,3 PGA and PGA in tissues:

Substance Concentration in cells (M)

PGK 130
GAPDH 1400
1,3 PGA 50
3PGA 200

The significance of these data are (i) glycolytic enzymes are present at quite high concentrations so
complexes between them may form (ii) the concentration of enzymes and their substrates are roughly
similar so there may be no free metabolites in solution (the derivation of the Michaelis-Menten equation in
enzyme kinetics, for example, assumes dilute solutions of enzymes and much higher concentrations of
substrate than enzyme. The assumption that the enzyme is saturated with substrate and most substrate is
unbound, is at odds with the situation with PGK and GAPDH)

There is some circumstantial evidence that at least in some systems that Triose Phosphate Isomerase (the
preceding enzyme in glycolysis) may form a complex with PGK, such that the substrate is passed between
them. For instance in the hyperthermophilic bacteria Thermotoga maritime both genes are fused and
expressed in the same polypeptide. One wonders, though, if this was such a great innovation, why don’t we
see it more often. Perhaps the report is just some genomic aretfact. Although the idea has been around for
some time that complexes of glycolytic enzymes may form into “metabolons”, there is really little hard
evidence. It is perhaps sufficient for us to appreciate the advantage of such complexes if they did exist. But
a clever hypothesis does not necessarily have to be true.

"Alice laughed: "There's no use trying," she said; "one can't believe impossible
things."
"I daresay you haven't had much practice," said the Queen. "When I was
younger, I always did it for half an hour a day. Why, sometimes I've believed
as many as six impossible things before breakfast."

The following is a review.

Menard L, Maugham D, and Vigoreaux J 2014 644 The Structural and Functional Coordination of
Glycolytic Enzymes in Muscle: Evidence of a Metabolon? Biology (Basel) 3, 623-644

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Summary
 Enzymes act as molecular jigs to align substrates precisely so that the reaction is faster and more
specific.
 In PGK ADP is precisely located to attack in-line the leaving phosphate of 1,3 PGA.
 PGK stabilises a high-energy penta-covalent intermediate with positively charged protein side-chains.
 On binding of both substrates there is a conformational change that brings relevant groups close
together and excludes water to prevent hydrolysis, which would otherwise lose the transferred
phosphate group to inorganic phosphate rather than retained as a high-energy bond in ATP.
 GAPDH and PGK enzymes are present at as high (or higher concentrations) than their substrates. Thus
the products of one may be passed quickly to the other with only a transient time in solution, which
avoids non-productive hydrolysis.

Appendix

Measuring the forward reaction

The basis for measuring the forward reaction is that
1,3PGA releases inorganic phosphate in acid
conditions whereas the other components do not.
Inorganic phosphate can be measured by a
subsequent colourimetric assay. The instrument used
is called a “stopped flow” apparatus. The reactants
(left) are forced into a mixer using motor driven
syringes. The reaction is allowed to develop and then
stopped after a time has elapsed by driving acid into
the mixer using another motorised syringe. Separate
runs are needed to collect a series of time points. At
each the amount of inorganic phosphate released is
measured. This decreases as the reaction goes
forward since there is less acid labile 1,3PGA and
more acid stable 3PGA.

Measuring the reverse reaction

The previous enzyme in the glycolytic pathway can

be coupled to a PGK catalysed reaction. The latter is
slower and so is rate limiting i.e. the rate of GAPDH
reaction is a measure of PGK reaction. In the reverse
direction GADPH catalyses the conversion of
NADH, which has a strong absorbance at 340 nm, to
NAD+, which has little absorbance. Thus the rate of
decrease of A340 signal is a measure of the rate of
PGK reaction in the reverse direction.

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Abbreviations
Abbreviation Full name
PGK Phosphoglycerate kinase
GAPDH Glyceraldehye Phosphate Dehydrogenase
3PGA 3-phosphoglyceric acid
1,3 PGA 1,3-phosphoglyceric acid
ATP adenosine monophosphate
ADP adenosine triphosphate